Chapter 1
An Introduction to Enzyme Science
Enzymes are astonishing catalysts – often achieving rate reaction) is reasonably fast – as is the case for the reversible enhancement factors1 of 1,000,000,000,000,000,000! hydration of carbon dioxide to form bicarbonate anion or for Water, electrolytes, physiologic pH, ambient pressure and the spontaneous hydrolysis of many lactones – an enzyme temperature all conspire to suppress chemical reactivity to (in this case, carbonic anhydrase) is required to assure that such a great extent that even many metabolites as thermo- the reaction’s pace is compatible with efficient metabolism dynamically unstable as ATP (DGhydrolysis z 40 kJ/mol) under the full range of conditions experienced by that and acetyl-phosphate (DGhydrolysis z 60 kJ/mol) are inert enzyme. Most enzymes also exhibit rate-saturation kinetics, under normal physiologic conditions. Put simply, metabo- meaning that velocity ramps linearly when the substrate lism would be impossibly slow without enzymes, and Life, concentration is below the Michaelis constant, and reaches as we know it, would be unsustainable.2 As a consequence, maximal activity when the substrate is present at a con- enzymes are virtual on/off- switches, with efficient centration that is 10–20 times the value of the Michaelis conversion to products in an enzyme’s presence and constant. In this respect, an enzyme’s action is more akin to extremely low or no substrate reactivity in an enzyme’s a variable-voltage rheostat than a simple on/off switch. absence. At millimolar concentrations of glucose and Biochemists recognize that substrate specificity is MgATP2 , for example, substantial phosphorylation of another fundamental biotic strategy for effectively organ- glucose would require hundreds to thousands of years in the izing biochemical reactions into metabolic pathways. Two absence of hexokinase, but only seconds at cellular analogous chemical reactions can take place within the concentrations of this phosphoryl transfer enzyme. Without same (or adjoining) subcellular compartments simply hexokinase, there would also be no way to assure exclusive because their respective enzymes show substrate or cofactor phosphorylation at the C-6 hydroxymethyl group. And even specificity directing metabolic intermediates to and through when an uncatalyzed reaction (termed the reference their respective pathways, often without any need for sub- cellular co-localization or enzyme-to-enzyme channeling. Substrate specificity also minimizes formation of unwanted, and potentially harmful, by-products. By controlling the 1 Catalytic rate enhancement (symbolized here as 3) equals the unit-less 1 relative concentrations of such enzymes, cells also avoid ratio kcat/kref, where the catalytic rate constant kcat (units ¼ s ) is the catalytic frequency (i.e., the number of catalytic cycles per second per undesirable kinetic bottlenecks or the undue accumulation 1 3 enzyme active site), and kref (units ¼ s ) is the corresponding of pathway intermediates. Experience tells us that first-order rate constant for the uncatalyzed reaction. The value of 3 will extremely reactive chemical species can also be sequestered be a direct measure of catalytic proficiency (i.e., an enzyme’s ability to within the active sites of those enzymes requiring their enhance substrate reactivity), if and only if the enzymatic and nonenzymatic reactions operate by the very same chemical mechanism, in which case the nonenzymatic reaction is called the reference reaction. Note also that the value of 3 achieved by any given enzyme need only be sufficient to assure unimpeded metabolism. In the 3 The term intermediate has several distinctly different meanings in Principle of Natural Selection, mutation is the underlying search biochemistry. In the context of the above sentence, intermediate refers algorithm for evolution, and any mutation that markedly improves 3 to a chemical substance that is produced by an enzyme reaction within beyond that needed for an organism’s survival should be inherently a metabolic pathway (A / B / C / P / Q / R, where B, C, P, unstable and subject to reduction over time. and Q are metabolic intermediates) and is likewise a substrate in 2 The upper limit on the room temperature rate constant for nonenzymatic a subsequent enzyme-catalyzed reaction in that or another pathway. In water attack on a phosphodiester anion, for example, is about 10 15 s 1, the very next sentence, intermediate refers to a enzyme-bound substrate, necessitating 100-million year period for uncatalyzed P–O cleavage enzyme-bound reactive species, or enzyme-bound product formed z (Schroeder et al., 2006). Depending on reaction conditions, the during the catalysis (E þ S # ES1 # ES2 # EX # EP1 # EP2 # z corresponding rate constant for hydrolysis of the b g P–O bond in E þ P, where ES1,ES2,EX,EP1, and EP2 are various enzyme-bound MgATP2 is around 10 4 to 10 6 s 1, and given that bimolecular species/intermediates) in a single enzymatic reaction. For reactions processes obey the simple rate law v ¼ k[A][B], rates for phosphoryl occurring in the absence of a catalyst, chemists routinely use the term 2 group transfer reactions (e.g., MgATP þ Acceptor # Phosphoryl intermediate to describe any reactive species Xi-1, formed during the Acceptor þ MgADP) would be suppressed even further at low course of chemical transformation, whether formed reversibly (i.e., # # / / micromolar-to-millimolar concentrations of acceptor substrates within Xi-1 Xi Xiþ1) or irreversibly (i.e., Xi-1 Xi Xiþ1). All such most cells. usages of intermediacy connote metastability and/or a transient nature.
Enzyme Kinetics Copyright Ó 2010, by Elsevier Inc. All rights of reproduction in any form reserved. 1 2 Enzyme Kinetics formation, while hindering undesirable side-reactions that The creation of organizationally complex neural networks, as would otherwise prove to be toxic. So enzyme catalysis is facilitated by the capacity of single neuronal cells to engage inherently tidy. Enzyme active sites can also harbor metal in tens of thousands of cell–cell interactions with other ions that attain unusually reactive oxidation states that rarely neurons via synapse formation, is also thought to underlie form in aqueous medium and even less often in the absence what we sense as our own consciousness. And at all such of side-reactions. The resilience of living organisms stems in levels, enzyme catalysis and control are inevitably needed for large measure from the capacity of enzymes to specifically or effective intracellular and intercellular communication. selectively bind other ligands (e.g., coenzymes, cofactors, As the essential actuators of metabolism, enzymes are activators, inhibitors, protons and metal ions). often altered conformationally via biospecific binding Attesting to the significance of enzyme stereospecificity in interactions with substrates and/or regulatory molecules the biotic world is that most metabolites and natural products (known as modulators or effectors) to achieve optimal contain one or more asymmetric carbon atoms. The stereo- metabolic control. An additional feature is the capacity of specific action of enzymes is the consequence of the fact that multi-subunit enzymes to exhibit cooperativity (i.e., both protein and nucleic acid enzymes are polymers of enhanced or suppressed ligand binding as a consequence of asymmetric units, making resultant enzymes intrinsically inter-subunit cross-talk). Because enzyme structure changes asymmetric. It should be obvious that any L-amino acid- can be triggered by changes in the concentrations of containing polypeptide having even a single D-amino acid numerous ligands, enzymes possess an innate capacity to residue cannot adopt the same three-dimensional structure as integrate diverse input signals, thereby generating the most a natural polypeptide. Although some enzymes utilize both appropriate changes in catalytic activity. An interaction is enantiomers of a substrate (e.g., glutamine synthetase is said to be allosteric if binding of a low-molecular weight almost equally active on D-glutamate and L-glutamate), substance results in a metabolically significant conforma- proteins containing exclusively L-amino acids are produced tional change. In most cases, modulating effects are nega- by the ribosome’s peptide-synthesizing machinery. This tive (i.e., they result in inhibition), but positive effects (i.e., outcome is the result of the stereospecificity of aminoacyl- those resulting in activation) are also known. Feedback tRNA synthases that supply ribosomes with activated regulation has proven to be a highly effective strategy for subunits, the stereochemical requirements of peptide controlling the rates of metabolic processes. When present synthesis, as well as ubiquitinylating enzymes and protea- at sufficient concentration, a downstream pathway inter- somes that respectively recognize and hydrolyze wrongly mediate or product (known as a feedback inhibitor) alters folded proteins. Cells also produce a range of enzymes, such the structure of its target enzyme to the extent that the as D-amino acid oxidase (Reaction: D-Amino Acid þ O2 þ inhibited enzyme exhibits little ot no activity (Scheme 1.1). H2O # 2-Oxo Acid þ NH3 þ H2O2), that remove certain Target enzymes (shown below in red) are most often posi- enantiomers (in this case, D-amino acids) from cells. In the tioned at the first committed step within a pathway or at case of protein enzymes, certain aspartate residues are also a branch point (or node) connecting two or more pathways. susceptible to spontaneous racemization as well as N-to-O The lead reactions are frequently highly favorable (DG << acyl shifts, and cells produce enzymes that recognize and 0), whereas the intervening reactions are generally revers- mediate the repair or destruction of proteins containing ible (DG ¼ 0), or nearly so (DG z 0). monomers having improper stereochemistry. Additional metabolic pathway stability is afforded by steady-state fluxes that resist sudden changes in rate or EEF F G H reactant concentrations. The processes lead to the phenom- E EF EG A A B C D E enon of homeostasis, wherein reactant concentrations appear EB EC ED EI EJ to be time invariant merely because the processes producing EEI I J K and destroying these reactants are so exquisitely controlled. In some respects, the behavior of the whole of metabolism Scheme 1.1 appears to exceed the sum of behaviors of its individual reactions. Experience has shown that hierarchically complex, large-scale networks often give rise to emergent properties Feedback inhibition (shown in blue) of target enzymes (i.e., properties of a highly integrated metabolic or physio- therefore precludes unnecessary accumulation of possibly logic system that are not easily predicted from the analysis of toxic metabolic pathway intermediates. By contrast, elevated individual components). Beyond the coordinated operation metabolic throughput (or flux)isobservedwhenanenzyme and regulation of the many pathways comprising interme- responds to an allosteric activator. In the latter case, the diary metabolism, other emergent properties of living enzyme achieves no or partial catalytic activity in the absence systems are evident in the adaptive resilience of signal of an activator, and biospecific binding of the activator alters transduction, long-range actions affecting chromosomal the target enzyme’s conformation in a way that increases its organization, as well as cellular morphogenesis and motility. catalytic efficiency. Although the hallmark of allosteric Chapter j 1 An Introduction to Enzyme Science 3 enzymes is cooperativity (i.e., subunit–subunit interactions forced to glean information haphazardly. A more effective altering the apparent substrate binding affinity), metabolic strategy starts with a reliable assay of catalytic activity and control is also achieved by the regulated synthesis and requires the experimenter to use this assay in the isolation of degradation of specific enzymes, by interconversion between the enzyme of interest from other contaminants (e.g., enzyme activity states via enzyme-catalyzed covalent modi- proteins, solutes, etc.) affecting the enzyme’s activity. In fication, by effector molecule mediated signal amplification, practice, absolute purity is not required as long as other and in some instances by substrate channeling. contaminating enzymes and proteins are without effect on Molecular life scientists have uncovered countless the enzyme of interest. It is helpful to apply the principles of instances wherein improper catalysis and/or regulation of organic chemistry to infer likely chemical transformations even a single enzyme reaction can greatly distress a living occurring during catalysis, using literature precedents to organism. Such mutant enzymes wreak havoc on cellular guide one’s thoughts about the roles of coenzymes and physiology. In fact, animal and plant diseases frequently cofactors and to focus on probable reaction intermediates. arise from point mutations that result in site-specific Ultimately, however, it is necessary to test whether each substitution of a single amino acid residue in an enzyme. reaction step occurs on a time-scale consistent with its role Elaborate proofreading mechanisms permit replication, in catalysis. This latter pursuit, called enzyme kinetics, transcription, and translation to proceed at rapid rates, while combines an interest analyzing temporal aspects of enzyme minimizing error propagation, and a battery of repair catalysis with the principles of physical chemistry and enzymes correct DNA damage arising unavoidably from quantitative rigor of analytical chemistry. photolysis, oxidation, alkylation, hydrolysis, and racemi- Some of the stages in the characterization of a complete zation. The same is true of errors occurring during the enzyme mechanism are listed in Fig. 1.1. Because initial-rate synthesis, splicing, and turnover of RNA transcripts. Ribo- kinetics is a relatively straightforward tool for analyzing somes must also occasionally commit errors, but with the enzyme catalysis, we may regard such experimental possible exception of prion protein formation the impact of approaches as the first stage in the systematic characteriza- low-level occurrence of ‘‘translational mutations’’ is apt to tion of an enzyme of interest. Pursuit of subsequent stages be minimal. Other more injurious mistakes made during depends on the objectives of the particular investigation. replication and transcription are known to culminate in This reference explains how enzyme kineticists formulate enzyme over-/under-production, defective regulation, and test models to: (a) explain the reactivity and energetics impaired stability, incorrect post-translational modification, of enzyme processes; (b) gain the most complete description improper subcellular targeting and compartmentalization, defective turnover, etc. A notable example is amyotrophic Stage-1: Initial Rate Kinetics lateral sclerosis or ALS (widely known as Lou Gehrig’s v versus [substrate(s)] → Km,Vm & VmIKm disease). This devastating neurodegenerative disorder is Substrate Specificity & Side-Reactions linked to the impaired action of superoxide dismutase; over- Product Inhibition → Substrate Binding Order accumulation of superoxide (O ) damages neurons, an Competitive Inhibition → Substrate Binding Order 2 pH Kinetics → pK’s of Catalytic Groups injury that is attended by profound pathological sequelae. Site-Directed Mutagenesis Another example is the discovery that Pin1-catalyzed cis- Stage-2: Chemical Studies trans prolyl residues isomerization can alter the structure of Determination of Reaction Stereochemistry the microtubule-associated protein Tau in axons and that Detection of Tightly Bound Coenzymes & Metal Ions Pin1 gene knockouts bring about progressive age dependent Detection of Covalent Intermediates Identification of Active-Site Residues by Affinity Labeling neuropathy characterized by motor and behavioral deficits, Stage-3: Isotope Kinetics attended by hyper-phosphorylation of Tau, as well as Tau Partial Exchange Reaction → Substrate Binding Order polymerization into neurodegenerative paired helical fila- Isotope Exchange at Equilibrium → Substrate Binding Order ments (Liou et al., 2003). Although more research is Isotope Trapping & Partition Kinetics → “Stickiness” required to assess the significance of such findings to the Positional Isotope Exchange → Reaction Intermediates Kinetic Isotope Effects → Reaction Intermediates onset of Alzheimer’s disease, it is already clear that reduced Stage-4: Fast Reaction Kinetics prolyl cis-trans isomerization activity can profoundly Continuous, Stopped-Flow & Mix/Quench Techniques impair neuronal function. Temperature-Jump & Pressure-Jump Techniques Enzyme chemists investigate biological catalysis by Stage-5: Single-Molecule Reactions assessing the structural and energetic features of the Reaction Trajectories elementary reactions comprising a multi-step enzyme Mechanochemistry of Force Generation mechanism. They seek to understand how activators and inhibitors alter the energetics of catalytic reaction cycles to FIGURE 1.1 Kinetic tools in modern enzyme science. Depicted here are the typical stages in order of complexity for the characterization of bring about effective metabolic regulation. The daunting an enzyme-catalyzed reaction. Within each stage are various experimental task of determining how an enzyme operates is never an approaches that will be discussed in detail in later chapters. Very few easy matter, and without a systematic approach, one is enzymes have actually been exhaustively investigated at all five stages. 4 Enzyme Kinetics of catalysis; and (c) understand how an enzyme’s regulatory or a mechanically generated force, etc.), space (e.g., posi- interactions affect the catalytic reaction cycle. Ideally, one tionally defined parameters x, y, z in Cartesian coordinates should consider as many reasonable models as possible for or r in fields), and time expressed in seconds. In chemical the reaction/process of interest. These rival kinetic models kinetics, we analyze the dependence of reaction rate on the should be as simple as possible: when stripped down to the concentrations of reactant(s), and although kinetic isotope bare essentials, any failure of a model to account for an effects depend on the masses of nuclei at or near the reaction experimentally determined property of the system becomes center, we are mainly concerned with electronic rear- sufficient justification for outright rejection of that model or rangements in molecules, as reflected by the nature of the for modifying it to account for other by essential properties/ chemical bonds within reactants, intermediates, and prod- interactions. Simplicity, precision, and generativity – these ucts. (Reaction rate is defined by the product of a reaction are the inherent virtues of highly effective models. rate constant and its reactant concentration(s), and for Simplicity demands that a system’s known properties are stochastic kinetic approaches, probabilities are often used in represented by the least number of components and/or place of macroscopic variables.) In chemical dynamics, the interactions. Precision requires explicit presentation of all main goal is to depict how the potential energy changes as required interactions, thus providing an opportunity to one varies the relative coordinates and momenta of the distinguish testable model-specific characteristics of rival atomic nuclei involved in the reaction (Polanyi and models. Generativity implies that the model should facili- Schreiber, 1974). The latter most often entails the applica- tate hypothesis-driven experimentation to test newly pre- tion of classical scattering theory relying on classical dicted properties in a recursive manner that stimulates new collision theory, with solution of the appropriate equations rounds of experimentation. Put plainly, a model is not worth for atomic and molecular motions as reactants proceed much, unless it fosters the formulation of new hypotheses along a trajectory on the potential energy surface. At the that spur additional rounds of experimentation. single molecule level, population-averaged parameters X Modern molecular life scientists have become, for want of give way to probabilistic expectations
to show that the rate of this reaction is linearly dependent favor of ammonia (Reaction: N2 þ 3H2 ¼ 2NH3). That on the concentration of sugar. Berthelot (1862) and process – now bearing Haber’s name – has forever altered Berthelot and de Saint-Gilles (1862) reached the same the human condition by augmenting Nature’s output of conclusion from studies on ethyl acetate hydrolysis, and ‘‘fixed’’ nitrogen by some 20–30%. To improve crop such observations led Guldberg and Waage (1867; 1979) to yields, farmers routinely inject synthetic ammonia postulate that chemical reactions must be highly dynamic, directly into the soil. with reactants and products relentlessly interconverting In considering the nature of a catalytic cycle, one may into each other, even at equilibrium. In advancing this take the case of heterogeneous catalytic decomposition of principle, widely known as the Law of Mass Action,they the toxic atmospheric pollutant N2O within the catalytic suggested that the rate in each direction of a reversible converter of a modern automobile. The cycle begins with reaction depends on reactant concentration (often chemisorption of N2O onto the platinum/palladium cata- expressed as the intensive variable molarity) and not the lyst, a step that weakens the bonds stabilizing nitric oxide, amount of substance (commonly given by the extensive to the effect that the N–O bond can dissociate. The latter variable mole). produces N2, which desorbs from the surface, leaving As discussed at length in Chapter 3, the modern oxygen radicals on the catalyst. At the catalyst’s operating conceptual framework for the discipline known as temperature, these radicals diffuse along the metallic chemical kinetics was founded late in the nineteenth surface until two of them encounter each other and century by the powerfully insightful contributions of the combine to form O2, the latter then desorbing from the Swedish chemist Svante Arrhenius and the German surface. French Chemist Paul Sabatier (Nobel Laureate in chemist Jacob van’t Hoff, who both became Nobel 1912) is credited with a principle bearing his name. Stated Laureates in chemistry. They and German physical in its simplest form, the Sabatier Principle asserts that for chemist Wilhelm Ostwald, the Nobelist credited for first effective catalysis, substrates and products must bind expressing reaction velocity as a change in reactant sufficiently tightly, so as to promote catalysis, but not too concentration per unit time (i.e., v ¼ d[Reactant]/dt), tightly so to prevent catalysis. Sabatier stressed the established the enduring concept that catalysts promote momentary nature of catalytic intermediates, a point that reactivity without altering the equilibrium position of the underscores their celerity and the importance of kinetics in overall chemical reaction. These investigators recognized analyzing their nature. that thermodynamics constrains catalysis: after each As discussed below, catalytic selectivity/specificity catalytic round, the catalyst releases its product and also allows chemists to control the stereochemical therefore cannot exert any cumulative effect on the outcome of reactions that would be otherwise nonspe- reaction’s standard Gibbs free energy change DG .This cific. And while organic chemistry of the 1950s relied on discovery increased the determination of chemists to just a few catalysts (mainly Hþ,OH ,Al3þ,Fe3þ,as discover catalytic substances and even to design artificial well as elemental Pt, Ni, and Pd) that were almost catalysts endowed with special properties. Speed and invariably stereochemically unselective, modern organic yield are the essence of catalysis, but the idea that one chemists have exploited a much wider repertoire of may impart reactivity to otherwise unreactive substances metallo-catalysts. lies at the heart of modern chemical enterprises. Nowhere Recognizing that all reactions proceed through the is this more evident than in the work of Fritz Haber, the formation and turnover of transition-state intermedi- notorious German chemical engineer5 and Nobel ate(s), one may consider the conversion of reactant A Laureate. Haber’s research team overcame the virtual into product P in the absence and presence of catalyst C. inertness of dinitrogen by carrying out some 20,000 In the uncatalyzed case, reactant A isomerizes through experiments, utilizing thousands of catalyst preparations a succession of intermediates and transiently reaches the under a wide range of reaction conditions. They eventu- activated complex Xz. As the least stable intermediate, ally settled on the use of iron filings to catalyze ammonia Xz exhibits an equal likelihood of reconverting to the synthesis from N2 and H2 at high temperature (600–800 reactant or going onward to product, such that the system K) and extreme pressure (300 atm). High temperature eventually reaches thermodynamic equilibrium. In the facilitated dissociation of highly stable bonds within N2 catalyzed reaction, reactant A first combines with cata- and H2, and pressure displaced the reaction equilibrium in lyst C to form the C$A, which then passes through a series of intermediates (e.g.,C$X1,C$X2, etc.) to reach C$Xz. As was true for the uncatalyzed process, the intermediate C$Xz can either return to C$A or advance to 5 During World War I, Haber supervised firsthand battlefield tests on the C$P, with product-release subsequently regenerating the efficacy of chemical warfare agents that later proved to irreversibly inhibit the enzyme acetylcholine esterase. Such activities would be catalyst. Michael Polanyi, father of Nobel chemistry subject to prosecution under the international treaties on war crimes laureate John Polanyi, was arguably the first to articulate signed at the close of that war. the notion that stabilization of the reaction transition-state Chapter j 1 An Introduction to Enzyme Science 7
Xz as the complex of catalyst and transition-state C$Xz 1.1.2 Synthetic Catalysts should greatly increase the forward and reverse reaction Chemists have created powerful catalysts that facilitate rates. The enhancement factor 3 (equal to vcat/vuncat) therefore applies both to the forward and reverse reac- chemical transformations in chemistry laboratories, oil tions, and a reaction’s equilibrium constant can be refineries, and even automotive exhaust systems. Table 1.1 expressed as: summarizes some of the most widely used catalysts that contribute to the trillion dollar petrochemical and agri- 3 vuncatalyzed vuncatalyzed chemical industry worldwide. Although metallic platinum, K ¼ reverse ¼ reverse ¼ Keq 1.1 3 vuncatalyzed vuncatalyzed palladium and nickel are constituents in many catalysts, the active forms consist of small surface imperfections, or step Autocatalysis is a special case of chemical catalysis in defects, and not merely the projected geometry of the metal’s which the active catalyst is also a product. An example is the internal crystal surface. These agents are often called formation of pepsin on its storage form pepsinogen in acidic heterogeneous catalysts, a term that indicates the presence of gastric juices: two phases: gaseous or liquid reactants binding and reacting on the surface of a solid catalyst. Catalysts, such as hydroxide ions and protons, which remain in the same phase as the reactants, are referred to as homogeneous catalysts. Initiating Reaction: Pepsinogen + H+ → Pepsin inact act Synthetic catalysts are often highly stable, allowing them to operate efficiently even in the face of elevated Autocatalytic Reaction: Pepsinogeninact + Pepsinact → 2 Pepsinact temperatures and pressures, as well as extremes of pH. Scheme 1.2 One unrelenting problem has been to design catalysts that resist fouling, or quenching, by tight-binding reaction products and/or metal ions. Most synthetic catalysts are where the inactive zymogen Pepsinogeninact is at first converted slowly by acid catalysis to the active enzyme also inferior to biological catalysts in at least four other respects, as they: (1) are less efficient at physiologic Pepsinact, it then rapidly catalyzes the conversion of any remaining zymogen to its active form. Note that temperature, low pressure, and neutral pH; (2) are rela- each catalytic round during the autocatalytic phase tively unselective; (3) rarely display sufficiently high doubles the amount of active enzyme until the concen- chiral recognition, a property that greatly limits their use tration of inactive enzyme is depleted (see Section 3.9.4: in preparing optically active biomolecules; and (4) are not Autocatalysis). regulated by feedback activators and/or inhibitors. Finally, the catalyst concentrations approach the concentrations of substrate(s) or product(s), the equilib- 1.1.2a Catalytic Hydrogenation rium position of the reaction, depending on the catalyst’s relative affinity for the reactant(s) and product(s). This The classical case of catalytic hydrogenation (Fig. 1.2) is effect can manifest itself in some rapid-mixing experi- a two-phase, or heterogeneous, process. The alkene or ments, particularly when reagent concentrations of enzyme alkyne is first adsorbed on the surface of the catalyst are utilized. alongside a dihydrogen molecule, whereupon the catalyst
TABLE 1.1 Selected Man-made Catalysts and the Reactions Catalyzed
Catalyst Category Process/Properties
Platinum-containing Chlorinated Alumina Heterogeneous Hydroisomerization (conversion of n-butane into isobutane) Platinum, Nickel, Palladium Heterogeneous Hydrogenation of double bonds # Iron Shavings/Dust Heterogeneous Haber ammonia process (Reaction:3H2 þ N2 2NH3) Silica/Alumina Zeolites; NiCoMo tri-metallics Heterogeneous Cracking of petroleum into volatile fuels Vanadium(V) Oxide, Palladium Heterogeneous Oxidation of exhaust from internal combustion engines Acids/Bases Heterogeneous/ Hydrolysis of carboxylic/phosphoric esters and anhydrides Homogeneous Grubbs and Hoyeyda Ruthenium Catalysts Heterogeneous Metathesis (see text for details) Chiral Catalysts Homogeneous Enantiomeric selectivity/specificity Catalytic Antibodies Homogeneous Over 100 different reaction types 8 Enzyme Kinetics
perfectly flat, and catalysis may be more effective in surface microenvironments. Another useful strategy is to deposit metal atoms onto other solid substrates that can greatly influence the resulting surface geometry and coverage. H H Given the great cost of metals like platinum, palladium H H and even nickel, chemists have attempted to maximize C C the active catalytic surface of metal catalysts. In such H H cases, platinum and palladium are combined with a char- coal support (also called the substratum or substrate). Raney nickel, for example, is a solid hydrogenation catalyst composed of fine grains of a nickel-aluminum alloy, and the catalyst known as ‘‘platinum on charcoal’’ consists of 5% platinum and 95% charcoal by weight. FIGURE 1.2 Schematic of catalytic hydrogenation of ethylene on Gold nanoparticles have also been employed as catalysts. a nickel, platinum, or palladium surface. In this idealized representa- To explain why ordinarily inert gold becomes a powerful tion, the metal surface acts as a rack, on which each of the reactants is catalyst, chemists have proposed that: (a) nanometer- stretched by binding to adjacent metal atoms. This physisorptive process sized particles contain many more surface dislocations occurs by interactions of reactant electrons with empty electron-deficient orbitals of the metal. Catalytic hydrogenation results from the heightened that serve as unusually reactive domains; (b) they are reactivity among the weakened intramolecular bonds of H–H and more or less electron dense than bulk gold; (c) such ] CH2 CH2, depicted above as dashed lines between reactant atoms. In particles contain numerous perimeter sites and/or many metals, step-like dislocations on the crystal surface are the actual ‘‘sticky’’ paracrystalline surfaces, and/or (d) nanometer sites of enhanced catalytic action. Except for the fact that heterogeneous sized particles have different metallic properties than catalysis occurs at the interface of a gas-solid, liquid-solid, or immiscible liquid-liquid phases, the process of catalytic hydrogenation resembles those of bulk gold (Bell, 2003). Other industrial catalysts a random bisubstrate enzyme-catalyzed reaction (i.e., reactants A and B include di- and poly-nuclear metal cluster complexes, add randomly to form an Enz$A$B, followed by conversion to product such as di-molybdenum and di-tungsten complexes, di- complex Enz$C, from which C desorbs from the active site to complete rhodium (II) complexes, as well as multinuclear Rh, Rh- the catalytic cycle). Co, and Ir-Co complexes. weakens their respective bonds and may even change the 1.1.2b Metathesis position and/or orientation of these bound species. The two hydrogen atoms then shift from their interactions with the The process known as olefin metathesis refers to position metal surface to the carbon atoms comprising a double or changing organochemical catalysis occurring in the triple bond, with attendant formation of a more saturated presence of suitable transition metal complexes, including hydrocarbon. The latter is more weakly adsorbed and soon various metal carbenes. These catalysts (particularly the departs from the catalyst’s surface. The exact nature and Grubbs Ruthenium Catalyst and Hoveyda Ruthenium timing of these events is still incompletely understood. Catalyst) facilitate bond-breaking and exchange of What is clear is that the metal surface acts as a rigid rack on substituents directly attached to the double bonds of the which the reactants are stretched to weaken the s-bond of coordinated olefins. A metal carbene initiates olefin H–H as well as the p-bond of an alkene (or alkyne), with the metathesis by reacting with an olefin to form a metal- effect that hydrogenation is facilitated. Because H–H and lated-cyclobutane intermediate, which then breaks apart R–C]C–R9 (or R–R9) bond lengths differ by ~0.5 A˚ , each to form a new olefin and a new metal carbene. This highly bond must be polarized to a different degree to reach the versatile chemical process can be used to: (a) swap optimal reaction transition-state. (A corollary is that the groups between two acyclic olefins (a process called likelihood of achieving this alignment in the catalyst’s cross-metathesis); (b) close large rings (ring-closing absence is extraordinarily low.) metathesis); (c) form dienes from cyclic and acyclic Classical transition metal catalysts, such as platinum, olefins (ring-opening metathesis); (d) polymerize cyclic palladium, nickel and rhodium, rely on their intrinsic olefins (ring-opening metathesis polymerization); and (e) inter-atomic spacing in their crystalline state or as multi- polymerize acyclic dienes (acyclic diene metathesis atom aggregates. That said, some catalysts actually rely polymerization). on step-dislocations on their roughened surfaces to create The commercial availability of these catalysts has the best sites for catalytic hydrogenation. To achieve greatly promoted the use of metathesis in macrolide higher catalytic rate enhancements, many synthetic cata- synthesis, where closure of large rings (i.e., those having lysts are deliberately designed to contain reactive surface ten or more atoms within them) is typically a low-yield defects or atomic dislocations. No metal surface is reaction. The power of olefin metathesis is that it Chapter j 1 An Introduction to Enzyme Science 9 transforms the –C]C– double bond, a functional group H C CH that is often unreactive toward many reagents. With certain 3 3 ] * O O catalysts, new –C C– double bonds are formed at or near O C H3C OH room temperature, even in aqueous media using starting * * N N * H C * OH materials that bear a variety of functional groups. Chemists 3 O C H C CH 3 C C 3 Yves Chauvin, Robert Grubbs and Richard Schrock shared * CH H C H3C 3 3 CH the 2005 Nobel Prize in chemistry ‘‘for the development of 3 the metathesis method in organic synthesis.’’ That these processes are highly relevant to the synthesis of enzyme TADDOLate bis(Oxazoline) inhibitors and therapeutic agents is illustrated by the use of tandem ring-closing metathesis to and subsequent hydro- genation to synthesis conformationally restricted cyclic dinucleotides joined with saturated connections between H * * H the nucleobase and the phosphate moieties (Borsting and N N O Nielsen, 2002). Metathesis also holds great promise for Co O O O O O industrial-scale reactions that are environmentally com- patible (so-called Green Chemistry). One concern, however, t-Bu t-Bu is the high cost, currently around $100 per millimol cata- lyst. Another is the use of toxic ruthenium, molybdenum, t-Bu t-Bu tantalum, etc. A third concern is the relative inefficiency of O O O O these catalysts, which typically operate at concentrations O Co O that are 2 10 mol-% of the reactant concentrations. N N H H * * 1.1.2c Chiral Catalysts
To mimic the remarkable enantiomeric preference exhibi- Salen Complex ted by many enzymes toward their chiral substrates, chemists have struggled to design homogeneous catalysts that are enantioselective. The trick is to introduce the right Bis(oxazoline) is loosely based on the structure of vitamin mix of binding energy, functional group chemistry, and one B12. Metal ion-containing catalysts, known as Salen or more chiral and/or dissymmetric sites (Yoon and complexes, facilitate epoxidation, epoxide ring-opening, Jacobsen, 2003). imine cyanation, and conjugate addition reactions. Such One such catalyst, known simply as TADDOLate compounds combine with reactive metal centers to produce ligand, is based on the structure of tartaric acid,6 one of the catalysts that effectively create asymmetric environments least expensive, naturally occurring chiral substances. that promote the selective binding and/or enhanced reac- (Asymmetric carbon atoms are indicated by asterisks.) tivity. As pointed out by Yoon and Jacobsen (2003), exactly TADDOL catalyzes aldehyde alkylation, ester acoholysis, what structural features account for the broad applicability of and iodo-lactonization. Another Diels-Alder catalyst synthetic chiral catalysts remains unclear. They suggest these
6Tartrate enjoys the distinction as the substance that Pasteur used to formulate his germinal ideas about stereochemistry as well as the first to have its absolute stereochemical configuration determined (Bijvoet, Peerdeman, and van Bommel, 1951). By verifying Emil Fischer’s fortuitous assignment for (þ)-glyceraldehyde (Rosanoff, 1906), there was no need to revise existing chemistry textbooks. COOH COOH COOH H OH H OH H C OH HOOC (R) H OH (R) CH HO C H 3 H OH HO H COOH H OH COOH COOH
Fischer Projection Fischer Projection Cahn-Ingold-Prelog Newman Projection
Shown above are various equivalent projections depicting the absolute stereochemical configuration of (þ)-tartrate. It is also worth noting that Pasteur (1858) reported the first stereospecific enzyme-catalyzed reaction, in which yeast fermented dextrarotatory tartaric acid, while leaving levorotatory tartaric acid completely intact. 10 Enzyme Kinetics catalysts possess rigid structures with multiple oxygen, charge. For example, were one interested in producing an nitrogen, and phosphorus atoms that allow them to interact antibody with the activity of a glycosidase, one might chose strongly with reactive metal centers. Because these agents a modified sugar that resembles the oxa-carbenium ion have a two-fold axis of symmetry, the number of possible intermediate with a half-chair conformation at or near the transition-state geometries is likely to be more limited. glycosyl carbon atom (see nucleoside hydrolase mechanism An inherent limitation in the design of chiral catalysts is the in Section 8.12.4; or lysozyme mechanism in Section 9.8.5c). current inability of chemists to reliably predict the type of The desired outcome is that the chosen hapten elicits anti- reactions that will be facilitated by a particular agent, the bodies that, when switched to bind on substrates, have the extent of its stereoselectivity, or the achievable catalytic rate capacity to facilitate the desired reaction. The candidate enhancement. For example, titanium complexes of chiral hapten is then coupled to a protein carrier, typically keyhole peptide-based Schiff’s base (or imine) ligands catalyze limpet hemocyanin (KLH), and the resulting conjugate is cyanation of epoxides, aldehydes, and imines with high used to immunize mice to produce one or more monoclonal enantioselectivity; the corresponding copper complexes antibodies. Catalytic antibodies are then identified on the catalyze allylic substitution of dialkyl-zinc nucleophiles; basis of their ability to catalyze the reaction of interest when whereas analogous zirconium complexes catalyze dialkyl- exposed to the desired substrate instead of the hapten (i.e., is zinc addition to imines (Josephson et al.,2001).Absent ‘‘switched’’). Because catalytic antibodies may not attain the a predictable outcome, one is left with the unenviable task of same stereospecificity as natural enzymes, and because surveying the reaction spectrum of each newly prepared catalytic antibodies may catalyze side-reactions, the exper- synthetic catalyst. The advent of High-Throughput Screening imenter is well advised to characterize the products with (HTS) promises to lessen the load of determining a catalyst’s respect to structure and enantiomeric purity. reactivity profile, but this approach remains to be perfected. In the case of ester hydrolysis, a phosphonate is a reasonably good isostere of the enzyme’s tetrahedral oxyanion transition state, with specificity determined in part 1.1.2d Catalytic Antibodies by the side chains R and R9. Among ‘‘semi-synthetic’’ catalysts listed in Table 1.1 are R' catalytic antibodies (also known as abzymes). These bio- R' OH O engineered proteins can be designed to accelerate specific O O organic chemical reactions. Basing his ideas on assertions C P about transition-state stabilization (Haldane, 1930; Pauling, R O R O 1946; Evans and Polanyi, 1936), Jencks (1969) succinctly advanced the following argument for catalytic antibodies: Transition-State Transition-State Structure Analogue If complementarity between the active site and the transi- tion state contributes significantly to enzymatic catalysis, One then raises monoclonal antibodies against the phos- it should be possible to synthesize an enzyme by construct- phonate-modified keyhole limpet hemocyanin (KLH). Alter- ing such an active site. One way to do this is to prepare an natively, one may select bacteria that express catalytic Fab’s antibody to a haptenic group, which resembles the transi- (i.e., antigen-binding fragments of antibodies) that are gener- tion state of a given reaction. The combining sites of ated by recombinant DNA methodology. Each antibody is then such antibodies should be complementary to the transition isolated and then evaluated for its ability to catalyze the state and should cause an acceleration by forcing bound hydrolysis reaction of interest. Observed rate enhancements substrates to resemble the transition state. should correlate with an antibody’s affinity for transition-state Because transition states are intrinsically unstable, analogue (TSA) versus reactant (R) (i.e., KTSA/KR,whereKTSA catalytic antibodies are selected by using chemically stable ¼ [Ab$TSA]/[Ab][TSA] and KR ¼ [Ab$R]/[Ab][R]). Experi- transition-state analogues used as immunogens. For mental results, however, often fail to satisfy the simplistic example, antibodies generated against a bent porphyrin ring assumption that the more closely an analogue resembles were found to catalyze the metallation of heme groups, a reaction transition state, the more effective is the antibody as presumably by straining the planar substrate toward a bent acatalyst. transition-state conformation. One inherent limitation in the use of transition-state In the classical ‘‘Bait-and-Switch’’ approach, one designs analogues to generate catalytically proficient antibodies is that a hapten (i.e., an immunogenic molecule that serves as the many interesting enzyme reactions are inevitably multi-step ‘‘bait’’) that structurally resembles a likely transition-state reactions, each with its own transition states. Therefore, no species (Pollack, Jacobs and Schultz, 1986; Tramontano, single analogue is likely to be an adequate template for each Janda and Lerner, 1986). In selecting the best haptens, one transition state. A second factor limiting the catalytic effi- focuses on key features of the transition-state intermediate, ciency of catalytic antibodies is the relative inflexibility of such as the arrangement of its atoms and/or its electrostatic most antibodies. While most enzymes are highly flexible and Chapter j 1 An Introduction to Enzyme Science 11 contain few internal disulfide bonds, the opposite is true of The observed rate enhancement 3 of 4,000,000 for this antibodies. A third limitation is that there is no easy way to catalytic antibody far exceeds the 103 to 105 values for increase the rate of product release in the design of catalytic others (Barbas et al., 1997), but falls short of aldolase antibodies. For most enzyme-catalyzed reactions, chemical (Reaction: Fructose-1,6 Bisphosphate # Glyceraldehyde- interconversion of enzyme-bound substrate and enzyme- 3-P þ Dihydroxyacetone-P) by 8 to 10 orders of magnitude. bound product is fast, and product release is frequently the A fortuitous case of an engineered antibody catalyzing rate-limiting step. The observed rate enhancements for a multi-stage transesterification reaction was reported by enzyme-catalyzed reactions therefore most often measure the Wirsching et al. (1995). This antibody behaved as a Ping Pong rates of product release. So increasing the rate of chemical enzyme (Catalytic Reactions:Eþ S1 # E$S1;E$S1 # F þ P1; interconversion of an antibody-bound substrate and antibody- F þ S2 # F$S2;F$S2 # E þ P2, where E and F are the free bound product may not do much to improve the observed rate enzyme and the acyl-enzyme, respectively). Evidence for multi- enhancements for antibody-catalyzed reactions. stage catalysis was adduced by the parallel-line patterns observed Reactive immunization is a new procedure for generating in a plot of 1/v versus [Ester] at several constant levels of the acyl- catalytic antibodies that tackles this problem by employing an acceptor alcohol (AAA) and in a plot of 1/v versus [AAA] at antigen that is so highly reactive that a chemical reaction occurs several constant levels of the ester. The resulting steady state in the antibody-combining site during immunization (Wirsching kinetic parameters were 3 and 7.3 mM, respectively, for the ester 1 et al., 1995). In the initial application of this approach, an and alcohol, and kcat was 21 min (the latter obviously much organophosphorus diester hapten was used as a ‘‘reactive slower that natural enzyme counterparts). The authors found that, immunogen.’’ A large number of the resulting antibodies cata- when a structurally related p-nitrophenyl ester was added to lyzed the formation and cleavage of phosphorylated intermedi- varying concentrations of the antibody with rapid mixing, equi- ates and subsequent ester hydrolysis. Wagner, Lerner and Barbas molar amounts of p-nitrophenol formed quickly, followed by (1995) applied the reactive immunization technique to generate a slower, steady-state release phase. The amplitude of the burst- antibodies that catalyze the aldol reaction. The mechanism for phase was proportional to the catalyst concentration. antibody catalysis of this reaction mimics that used by natural Other semi-synthetic enzymes have been prepared by Class-I aldolase enzymes. Immunization with a reactive modifying binding proteins and enzymes. For example, Zemal compound covalently trapped a Lys residue in the binding pocket (1987) observed catalysis of p-nitrophenylester hydrolysis of the antibody by formation of a stable vinylogous amide. The (enhancement factor 3 ¼ 1900) by heme-depleted myoglobin, reaction mechanism for the formation of the covalent antibody- a property that can be explained by the apolar binding pocket hapten complex was recruited to catalyze the aldol reaction. The with its two imidazoles that normally interacts with the heme. antibodies use the epsilon-amino group of Lys to form an Likewise, upon attachment of a flavin cofactor to Cys-25 enamine with ketone substrates and use this enamine as a nascent within papain’s active site, the resulting synthetic enzyme (or carbon nucleophile to attack the second substrate, an aldehyde, to synzyme) was found to catalyze oxidation of dihy- form a new carbon–carbon bond. Barbas et al. (1997) later dronicotinamide to nicotinamide with concomitant reduction designed additional antibody catalysts for aldol condensation of the flavin (Kaiser and Lawrence, 1984; Slama et al.,1984). reaction, based on the intermediates shown in Scheme 1.3. What becomes clear from model studies is that enzymes do much more than stabilize reaction transition states: they bind, orient, desolvate, and destabilize substrates; they push/pull O O H Enz protons to/from substrates, intermediates and products; they R1 N promote nucleophilic reactivity; and they exploit metal ions as
CH3 templates, as Lewis acids, and as highly reactive redox species CH3 HO R3 that are otherwise inaccessible in aqueous medium. Enzymes also exhibit a remarkable capacity to manage enthalpy and O R2 entropy changes throughout the catalytic reaction cycle, Substrate culminating in the release of reaction products. Although the most up-to-date approach uses a transition-state analogue to O generate the initial specificity, followed by site-directed Enz mutagenesis to provide essential catalytic groups, obtaining HN R1 catalytic antibodies is still hit-or-miss. Underscoring the limited rate enhancements achieved with R3 catalytic antibodies is the discovery that a so-called off-the-shelf CH O H 3 protein (bovine serum albumin) exhibits rate enhancements that R2 O rival tailor-made catalytic antibodies. Noting that Thorn et al. Product Intermediates (1995) described an antibody catalyzing the eliminative ring- opening of benzisoxazole, Hollfelder, Kirby and Tawfik (2001) Scheme 1.3 tested whether the lysine side-chain amines might also participate 12 Enzyme Kinetics in this general base-catalyzed reaction. With human albumin, shouldn’t be surprising. While Fritz Wo¨hler had succeeded 1 they obtained a kcat of 28.8 9.7 min , albeit with a prompt in synthesizing urea in 1826, the field of physical organic onset of product inhibition after only around 10 catalytic cycles. chemistry, which deals with the underlying kinetics and They also found that the rate enhancements reported for catalytic mechanisms of organochemical reactions, developed rela- antibodies depended on the somewhat arbitrary choice of solvent tively slowly until the late nineteenth century. What most conditions applied to the reference reaction. Until chemists can limited the progress in chemical kinetics of organic and increase the flexibility of catalytic antibodies, the ability to inorganic reactions was the lack of reliable methods for ‘‘teach’’synthetic catalysts and antibodies to mimic enzymes will quantifying changes in the concentration of reactants or remain an insuperable task. products. Spectrophotometers were nonexistent, because the then primitive electronic circuitry and low voltages 1.1.2e Synthetic Enzymes available from batteries precluded the fabrication of pho- Enzyme chemists have labored assiduously to fashion novel tomultiplier tubes. The need for a conveniently observable catalysts from structural proteins or to transform biospecific property led to early studies on the action of a crude ligand binding sites into active sites. Although the develop- preparation of emulsin on the hydrolysis of emulsified ment of crown ethers by Nobelist Donald Cram is often amygdalin, a sparingly soluble ester isolated from apricot erroneously credited as an early breakthrough in the synthesis pits. Emulsin later proved to be an enzyme that readily of artificial enzymes, U.S. chemists Myron Bender and Ronald converts the visibly milky white, aqueous suspension of Breslow pioneered these efforts. Bender and Breslow used amygdalin into transparent (water-soluble) products. With synthetic organic chemistry to introduce catalytically active that simple assay, the concept of catalysis could be substituents (e.g., chiefly carboxyl and imidazole groups) on demonstrated. It was, however, the advent of the polar- the rim of cavity containing cyclodextrins (see Section7.11for imeter that made possible the quantitative investigation of a discussion of cyclodextrin inclusion complexes). Before his how reaction rate depends on changes in the concentration early demise, the American chemist E. Thomas Kaiser had of optically active reactants or products. Even so, organic attempted to refashion the active sites of various heme proteins chemists lacked the means to synthesize chiral compounds, and a few enzymes to create synthetic enzymes with novel the latter being the sole province of physiologic chemistry catalytic properties. His creative efforts were met with modest (biochemistry). Because the degree of rotation of plane- progress toward the goal of fashioning new biocatalysts. The polarized light was a linear function of the molar monograph Artificial Enzymes edited by Breslow (2005) concentration of an optically active substance, this tech- presents a series of cogent reviews on artificial enzymes, nique provided the opportunity to demonstrate unambigu- ously that the acid-catalyzed hydrolysis of sucrose brought including biomimetic chemistry, vitamin B6-based enzyme models, synthetic polymers with enzymatic activity, catalytic about stereochemical inversion (i.e., a change in the antibodies, protein-based artificial enzymes, artificial metal- direction of rotation of plane polarized light. Likewise, the loenzymes, as well as artificial restriction enzymes. To date, corresponding action of the enzyme invertase (Reaction: # these efforts have met with uninspiring success, often for the Sucrose þ H2O D-Fructose þ D-Glucose) could be same reasons already noted above for catalytic antibodies. monitored reliably. The availability of polarimetry as a simple, highly sensitive, and reproducible quantitative technique essentially established chemical kinetics as 1.2 BIOLOGICAL CATALYSIS a rigorous physical science. 7 That the rates of enzyme-catalyzed reactions were studied 1.2.1 Roots of Enzyme Science long before corresponding organic chemical reactions The origins of enzymology as a scientific discipline can be traced to Spallanzani who, in 1783, demonstrated that 7 It is helpful to understand some basic terminology used by enzymologists. gastric juices liquefied meat, and to Gay-Lussac who, in A simple enzyme is a biological catalyst made wholly of protein, although 1810, reported that yeast growing anaerobically could more than one polypeptide chain may be part of the active enzyme. A ferment sugars into ethanol and CO2. Enzymes were first complex enzyme is composed of one or more polypeptide chains plus discovered in 1833 when Anselme Payen and Jean Persoz a low-molecular-weight organic molecule or metal ion at its active site. The term holoenzyme refers to the entire complex enzyme, whereas the found that an alcohol precipitate of malt extract contained term apoenzyme refers only to the protein component. If the non-protein the thermolabile substance diastase, which converted starch component binds non-covalently to the apoenzyme, it is called into sugar. Justus von Liebig proposed that fermentation and a coenzyme. (Many coenzymes contain structural elements of vitamins.) A digestive processes were inherently the result of chemical metal ion that binds directly to the protein is called a metal ion cofactor. action. In 1835, the German physiologist Theodor Schwann A prosthetic group is a relatively small organic molecule that is usually extremely tightly, or even covalently, bound. Some prosthetic groups also discovered that, in a manner similar to that of acid (as contain a metal ion (e.g., heme is protoporphyrin IX plus Fe(II) or Fe(III) discovered decades earlier by American physiologist John ion) held in place by coordinate covalent bonds. Young), gastric juice also contained its own digestive Chapter j 1 An Introduction to Enzyme Science 13 substance that became known as pepsin (from the Greek 1.2.2 Enzyme Technology pepsis for digestion). Work many years later established that pepsin is an enzyme. In many respects the forerunner of modern biotechnology, Given his remarkable chemical intuition and role as the field of enzyme technology was born in Copenhagen in a reductionist, it is remarkable that the great French 1874 with the establishment of the Christian Hansen’s chemist Louis Pasteur steadfastly adhered to the view that Laboratory. Although mainly focusing on the production of fermentation was uniquely the province of living yeast wax as a coating for cheeses, Hansen’s Laboratory became cells. His view supported the vitalists, who asserted that the first company to market a standardized preparation of life is the manifestation of a vital force (or, e´lan vital), the the enzyme rennet for use in cheese-making (Tauber, 1943). life-creating principle immanent in all living organisms. By controlling the rate and extent of milk curdling, The opposing mechanistic view that living systems would Hansen’s efforts greatly increased the quantity, quality, and inevitably be shown to obey the laws of chemistry and shelf life of European cheeses. While living in the United physics was held by the German physiologist William States in the early 1890s, the Japanese scientist Jokichi Ku¨hne, who in 1878 coined the phrase enzyme (from the Takamine developed a water–alcohol extraction method to Greek en and zyme, standing for ‘‘in’’ and ‘‘yeast’’) for isolate the powerful starch digesting enzyme Takadiastase. the fermentative substance in yeast. In 1893, the Latvian The latter was the trade name derived by combining ‘‘Taka’’ scientist Wilhelm Ostwald formally classified enzymes as from his name with ‘‘diastase,’’ the latter by then an already catalysts, even though their chemical nature was still well-known amylase preparation from the fungus widely debated (Ostwald, 1894). To explain the specific Aspergillus oryzae. His patent, granted as No. 525,823 by action of glycolyzing (i.e., sugar-cleaving) enzymes, Emil the U.S. Patent Office on September 11, 1894, was the first Fischer (1894) proposed his Lock-and-Key Hypothesis to teach proprietary aspects of enzyme technology. asserting that enzymes are rigid templates, into which Takamine’s efforts inspired what is now a century-old substrates must insert with the same high precision as Japanese tradition of using enzymes and highly controlled a key fitting into its corresponding lock. However, it was fermentation to improve production of sugars, cheese, another German chemist Eduard Bu¨chner, who in 1897 beer, vinegar, bread, fermented soy products, etc.,to proved that metabolism can take place outside intact produce fine chemicals like monosodium glutamate, ino- living cells. He innovated the procedure of grinding yeast sinic acid, and vitamins, and to isolate new drugs and anti- in abrasive sand, followed by passage through a paper metabolites. filter to obtain a cell-free extract. Noting the release of It is important to recognize that fermentation science and enzymology have profoundly altered the course of history. CO2 bubbles after adding the resulting extract to a sucrose solution, Bu¨chner correctly inferred that the extract itself A notable example is acetone-butanol fermentation. Pasteur acted as a catalyst, even in the absence of intact cells and was the first to identify butyric acid as a fermentation therefore any possibility of a vital force. The clean-cut metabolite, and acetone formation was later demonstrated result earned Bu¨chner the Nobel Prize in Chemistry, and by Schardinger (1905). In 1911, Fernbach and Weizmann the simplicity of his protocol ushered in the modern era of first reported on bacteria that produced amyl alcohol, systematic biochemical research. In 1898, Duclaux sug- ethanol, and acetone as stable metabolic end-products of gested that the suffix ‘‘-ase’’ be used in biochemical potato starch fermentation. A year later, Weizmann isolated nomenclature to distinguish enzymes from biological an organism that fermented all known starches and substances devoid of catalytic activity. produced acetone in much higher yield. Those were In his 1894 paper, Fischer asserted that among the desperate times, and sensing the significance of his agents that serve the living cell, the proteins are the most discovery in low-residue lacquers to waterproof cloth-sided important, but the mounting evidence that enzymes were airplanes as well as for explosives, the ardent Manchester proteins was stubbornly resisted by Richard Willsta¨tter. Zionist wrangled a promise (now known as the Balfour Having earned the Nobel Prize for working out the Declaration) that England would support his life-long goal structures of chlorophyll and heme, Willsta¨tter held that of returning Jews to Palestine. British reluctance to fulfill low-molecular-weight substances associated with proteins that promise led to the post World War II struggle that were the true catalytic entities. His view persisted until the ultimately established Israel, with Weizmann elected its first American scientist James B. Sumner (1926) crystallized president. Ironically, the Axis Powers relied on the immense urease, demonstrating that its catalytic power rested in the intellect of none other than Emil Fischer to manage the protein itself. Subsequent work by John H. Northrop German chemical industry during World War I. Failure of demonstrated that proteases could likewise be crystallized the Axis, loss of his two sons in that great war, and and that the protein was the sole component responsible advancing cancer overwhelmed Fischer, who committed for catalysis. The weight of their combined findings suicide in 1919. persuasively overwhelmed all doubters, and so doing Today, beyond the use of enzymes in biomedicine, earned them the Nobel Prize. enzyme technology (Tables 1.2 and 1.3) has expanded to 14 Enzyme Kinetics
TABLE 1.2 Some Commercial Applications of Enzyme Technologya
Product Enzyme application
Animal Feed Phytases hydrolyze abundant phytate (myo-inosital hexaphosphate) stores in plants used as animal feed, thereby increasing the nutritional value of the feed by releasing phosphate and bound metals from the phytate. Cheese-making Rennet cleaves k-casein between Phe-105 and Met-106, thereby destroying the latter’s ability to stabilize milk as a colloidal suspension, resulting in its calcium ion-induced coagulation into curd and liquid whey. (Treatment of soft cheeses with hen egg white lysozyme destroys Listeria monocytogenes, an infectious bacterial pathogen in those with compromised cell-mediated immunity.) Baking Industry Combined action of glucose oxidase and catalase removes glucose from egg whites prior to drying into dried egg white. Glucoamylase releases b-D-glucose from 1,4-a-, 1,6-a- and 1,3- a-linked glucans to yield high-glucose syrup. b-Amylases liberate maltose from barley starch in the production of high-maltose syrup. Invertase action on sucrose yields glucose and fructose, providing a sweeter syrup that is less apt to granulate than pure sucrose syrups. High-fructose Corn Syrup (HFCS) In this three-step process, Bacillus species a-amylase acts on cornstarch to produce shorter- chain polysaccharides, Aspergillus glucoamylase yields glucose, and glucoisomerase action increases fructose content to ~42%. Because HFCS is substantially sweeter than glucose or sucrose, less is required as a sweetener primarily in baked goods, candy, and soft drinks. Ethanol Production FermgenÔ protease is a proprietary fungal enzyme (pH optimum ¼ 3.0–4.5) that promises higher rates and yields of ethanol from fermentation for corn-, milo-, or wheat-based substrates by: (a) increasing availability of essential yeast nutrients in the form of amino acids, peptides and free amino nitrogen; and (b) hydrolyzing protein matrices within kernels, thereby facilitating use of otherwise hydrolysis-recalcitrant starches. Meat Tenderizing Papaya juice (rich in papain), pineapple juice (rich in bromelin), and orange juice (rich in ficin) are all highly effective tenderizers. (Processed papaya latex extract is sold under the brand name AccentÔ.) Fruit Juices, Wine, and Beer Combined action of Aspergillus pectinase and Monilia diastastase greatly reduces cloudiness, especially important for sparkling wines. In the absence of colloidal pectin, improved filtration/pressing also increases volume by 15–20%. Textiles Laccases (polyphenol oxidases) are used in the textile industry for dye bleaching in the production of ‘‘stone-washed’’ denim. Cellulases are sold to the textile industry for cotton softening and denim finishing. Alkaline pectinase, poly(vinyl alcohol)-degrading enzyme, cutinase and catalase are also used for cotton preparation. Pectinase and hemicellulases are used to soak and loosen bast (long and strong central) fibers for high-quality fabrics. Proteases remove contaminating proteins from silk fibers without effect on fibroin. Transglutaminase is used to introduce cross-links into wool, thereby strenthening fibroin strands. Amylases remove insoluble starchs and sizing from silk and cotton to improve quality of dyeing and printing processes. Tobacco Catalase reduces nicotine content. Glucosidases form the desired brown pigment by hydrolysis of quercitin-rhamnoglucoside (rutin). Amylases and invertases increase glucose and fructose content for improved taste. Leather Proteases (pepsin and trypsin as well as extracts of Aspergillus oryzae cultivated on rice, elastin, and keratin) remove flesh, blood and hair from fresh hides without affecting leather’s collagen network. Lipases remove oils that retard tanning and dyeing. Paper Production b-Xylanases are used in the treatment of paper pulp to reduce the use of chlorine for bleaching. Detergents Proteases (mainly subtilysin) remove proteins from food, skin, and saliva that accumulate on clothing. Haloperoxidases are now employed to generate ‘‘color safe’’ bleaches. These enzymes are often stabilized by intramolecular –S–S– linkages. More than half of all detergents now contain enzymes as a proprietary constituent. Sewage Treatment Lipases release enzymes from microbes to greatly accelerate the degradation of raw sewerage. Reducing Spread of Prion Diseases Residing deep within the fissures in the surfaces of stainless steel surgical devices, prions causing variant Creuzfeldt-Jakob Disease (vCJD) can resist standard sterilization procedures. PrionzymeÔ (a proprietary enzyme), the Bacillus-derived MSK103 protease, as well as a combination of proteinase K and pronase (the latter in the presence of SDS) can hydrolyze vCJD prions. These enzymes may therefore facilitate the sterilization of neurological and dental surgery instruments.
aThe interested reader should consult Tauber (1943) for detailed early accounts of the commercial utility of enzymes. Chaplin and Bucke (1990) present lucid descriptions of these and other more contemporary applications of enzymes in commerce. Chapter j 1 An Introduction to Enzyme Science 15
TABLE 1.3 Several Commercially Important Enzymesa
Type Enzymes
Carbohydrases a-Amylases; Alkaline a-Amylase; b-Amylase; Cellulase; Cyclodextrin glycosyl tranferase; Dextranase; a-Galactosidase; Glucoamylase; a-Glucosidase; Hemicellulase; Invertase; Lactase; Lysozyme; Naringanase; Pectinase; Pentosanase; Pullulanase; and Xylanase. Proteases Acid protease (Pepsin); Alkaline protease; Bromelain; Chymosin; Ficin; Neutral proteases (Trypsin, Chymotrypsin); Papain; Peptidases; Rennet; Rennin; Subtilisin; and Thermolysin. Lipases Triglyceridases and Phospholipases. Other hydrolases Amidases; Aminoacylase; Apyrase; Chlorophyllase; DNA restriction endonucleases (300þ enzymes); Feruloyl esterases; Glutaminase; Penicillin acylase; Phytase; Phosphatases; Pregastric esterases; and Ribonucleases. Oxidoreductases Amino acid oxidase; Catalase; Chloroperoxidase; Glucose oxidase; Glutathione peroxidase; Hydroxysteroid dehydrogenase; Laccase; Lactate dehydrogenase; Lipoxygenase; Lysyl hydroxylase; Lysyl oxidase; Peroxidase; Polyphenol oxidase; Sorbitol oxidase; Sulfhydryl Oxidase; and Xanthine oxidase. Decarboxylases Acetolactate decarboxylase; Aspartic b-decarboxylase. Polymerases RNA-dependent DNA polymerase (reverse transcriptase); Taq DNA polymerase; Vent DNA polymerase. Lyases Fumarase; Histidase. Isomerases Glucose isomerase; Xylose (Glucose) isomerase.
aThe interested reader should consult Tauber (1943) for detailed accounts of the commercial utility of enzymes. Chaplin and Bucke (1990) present lucid descriptions of these and other more contemporary applications of enzymes in commerce. include the use of enzymes in the production of foodstuffs, burgeoned over the years. Those interested in such appli- including hydrolysis of starch, production of glucose- and cations should consult Biocatalysts and Enzyme Technology maltose-rich syrups as well as high fructose corn starch, (Buchholz, Kasche and Bornscheuer, 2005). Another valu- derivation of glucose from cellulose, use of lactases in the able resource is Enzyme Catalysis in Organic Synthesis: A dairy industry, extended applications of enzymes in the Comprehensive Handbook (Drauz and Waldmann, 2002), preparation and storage of fruit juices, and improvement of which provides tried and true methods for using enzymes in wines, beers and distilled spirits and (Chaplin and Bucke, organic synthesis, a exhaustive table of all the commercially 1990). Enzyme technology has likewise improved produc- available enzymes, as well as comprehensive registers for tion of detergents, color-safe bleach, leather and wool. targeted searching according to enzyme, compound, or Modern biotechnology grew out of genetic engineering reaction type. in the early 1970s by the discovery of restriction enzymes by Daniel Nathans, Hamilton Smith and Werner Arber and 1.3 DEVELOPMENT OF ENZYME KINETICS the advent of recombinant DNA techniques, pioneered largely by Paul Berg, Herbert Boyer, and Stanley Cohen. The idea that an enzyme first combines with its substrate Nothing written here can adequately encapsulate the was suggested by Wurtz (1880), who found that papain momentous growth of biomedicine arising from recombi- appeared to form an insoluble compound with fibrin prior to nant DNA. In vitro protein synthesis promises to revolu- hydrolysis of the latter. O’Sullivan and Tompson (1890) tionize the production of pyrogen-free proteins, enzymes, reached a similar conclusion, based on their observation that and antibodies for use in highly specific and low-toxicity invertase is protected by its substrate sucrose against therapies. thermal denaturation. The theoretical basis of enzyme For many years, enzymes found limited application in kinetics was consolidated through the work of Adrian the organic chemistry laboratory. The notable exception was Brown (1892, 1902) and Victor Henri (1903), whose work the use of pig kidney acylase for the resolution of secondary on enzyme-substrate complex formation foreshadowed alcohols via stereoselective ester synthesis, followed by (‘‘adumbrated’’, as J. B. S. Haldane (1930) put it) the chromatography to separate the product. monumental paper by Leonor Michaelis and Maude Menten (1913). Their famous relationship (Eqn. 1.2) explains the kinetic behavior of literally thousands of enzyme-catalyzed OAc OH Esterase reactions.
CN CN Vm H C H C v ¼ 1.2 3 3 K 1 þ ½S Enzyme-mediated enantiomeric enrichment is discussed in greater detail in Section 5.10. The widespread utility of The Michaelis-Menten treatment is based on the rapid- enzymes in organic and pharmaceutical chemistry has now equilibrium assumption that the concentrations of free 16 Enzyme Kinetics
enzyme EF, free substrate SF, and enzyme-bound substrate peroxidase (Reaction: Leuko-malachite Green (colorless) þ E$X are defined thermodynamically: Kd ¼ [EF][SF]/[E$X]. H2O2 # Malachite Green (lmax ¼ 612 nm) þ 2H2O). John B. S. Haldane later introduced the concept of a steady- Chance also introduced the use of analogue (and later pio- state flux (e.g., d[E$X]/dt z 0) to enzyme kinetics and neered digital) computers for modeling the kinetic behavior metabolism (Briggs and Haldane, 1925; Haldane, 1930). of individual enzymes as well as those forming a metabolic Both approaches sample rate behavior over the course of pathway. Exactly how the Nobel Institute has failed to many catalytic reaction cycles.8 Haldane’s use of the recognize Chance’s enormous contributions to modern steady-state approximation pre-dated the development of chemistry is an enigma. non-equilibrium thermodynamic theory that now helps us to After the discovery of the phenomenon of nuclear comprehend the robust stability of steady states. magnetic resonance in 1946 by Bloch and Purcell, biolog- By the mid-nineteenth century, chemists Michael ical NMR spectroscopy was ushered in by Mildred Cohn Faraday and Antoine Lavoisier showed that all redox and others over the ensuing decades. Likewise, surging reactions (Overall Reaction:Aox þ Bred # Ared þ Box)can interest in sonar and shock-wave technology during World be treated as the sum of two half-reactions (Reduction Half- War II, coupled with the theory of pressure-induced Reaction:Aox þ e # Ared;andOxidation Half-Reaction:Bred chemical relaxation (Einstein, 1920) provided the impetus # Box þ e, where e represents an electron). This concept led for the investigation of individual steps (elementary reac- to the idea that other chemical processes may likewise be tions) within multi-step kinetic mechanisms. Fast reaction dissected kinetically into component (or elementary) methods, especially those pioneered by Nobel Laureates reactions. Manfred Eigen (temperature-jump technique), Ronald In 1910, the German electrochemist and Nobel Laureate G. W. Norrish (shock-tube and pressure-jump techniques) Walther Nernst extended Maxwell’s theory of gases by and George Porter (flash photolysis), completely revolu- suggesting that fast elementary steps in solution-phase tionized experimental chemical kinetics. reactions might be gainfully explored by chemical relaxa- Although somewhat beyond the current discussion, one tion techniques. However, instrumentation of suitable cannot minimize the impact of developments in physical stability and sufficient sensitivity was unavailable at that organic chemistry on the emergence of enzyme science. The time. Recognizing a need to probe the kinetics of hemo- British chemist Keith Ingold introduced the terms electro- globin oxygenation in much greater detail, Hamilton Har- phile for an electron-seeking functional group, nucleophile tridge and Francis Roughton (1923, 1926) introduced the for nucleus-seeking functional group, tautomerism for keto- rapid-mixing technique, known as continuous-flow, that enol isomeric rearrangements, and inductive effect to necessitated the use of 0.1–0.5 liter volumes of reactants. account for electronic effects of nearby entities on func- Britton Chance (1943) perfected their designs through his tional group reactivity. A fundamental advance was his ingenious design of a low-volume, stopped-flow rapid- conceptualization of the respective dissociative and asso- mixing device that used a spectrophotometer to detect and ciative features of SN1 and SN2 nucleophilic substitution analyze intermediates formed transiently by horse radish mechanisms at saturated carbon bonds. (Later work dis- closed that corresponding SN1 and SN2 mechanisms are also at play in phosphotransfer reactions.) Another Briton, Ronald Bell, connected the acid base theory of his mentor 8 Initial-rate enzyme experiments analyze multiple-turnover processes averaged over numerous catalytic turnovers. Multiple-turnover kinetic Brønsted to the origins of hydrogen isotope effects and phenomena are usually examined at low concentrations of enzyme, and correctly predicted that the kinetic isotope effect should be the accumulation or depletion of an enzyme-bound reactant species maximal when the proton is half-transferred in the reac- during the steady-state phase is assumed to be time invariant (i.e., tion’s transition state. Perhaps the most influential of Bell’s z D[EX]/dt 0). The number of turnovers occurring during an initial-rate contributions was his development and understanding of measurement equals D[P]/[EX] ¼ D[P]/{[P]t¼t [P]t¼0}, where [P] is the concentration of product formed, and [EX] is the concentration of quantum mechanical tunneling, or as he called it the tunnel enzyme-bound reactant over the period of measurement. The term correction for isotope effects involving proton (and hydride) single-turnover process refers to events occurring over one turnover or transfer processes. With their later keen interest in under- cycle of catalysis. As discussed in Chapter 10, single-turnover properties standing biological proton transfer, Bell’s disciples John are usually measured at high concentrations of enzyme using rapid Albery and Jeremy Knowles found warm acceptance of reaction techniques, such that the accumulation or depletion of an enzyme-bound reactant species, say EX, may be detected and quantified. their novel ideas on enzyme catalysis. Because the observed rate is a population average for many molecules Over the past half-century, enzyme kinetics has matured undergoing a single-turnover, the rate constants obtained are likewise into a highly sophisticated and innovative discipline. average values. The term single-molecule kinetic process refers to events Although the current state of any field is the sum of contri- occurring at the level of individual enzyme molecules undergoing one or butions of countless investigators, the following scientists more catalytic reaction cycles, observed by a suitable high-sensitivity microscopical technique. As discussed in Chapter 12, one can also study made advances so notable that they personify the field: reactions at the single-molecule by measuring local accumulation of Robert Abeles – Mechanism-based inhibitor design; product molecules generated by spatially isolated enzyme molecules. Cobalamin-dependent reactions; Robert Alberty –pH Chapter j 1 An Introduction to Enzyme Science 17
TABLE 1.4 Nobel Prizes Awarded for Research in Enzyme Sciencea
Year Laureate Award Cited achievement
2009 Venkatraman Ramakrishnan Chemistry Ribosome structure and mechanism 2009 Thomas A. Steitz Chemistry Ribosome structure and mechanism 2009 Ada E. Yonath Chemistry Ribosome structure and mechanism 2006 Roger Kornberg Chemistry Mechanism of transcription (RNA polymerase) 2004 Aaron Ciechanover Chemistry Mechanism of enzymatic ubiquitination 2004 Avram Hershko Chemistry Mechanism of enzymatic ubiquitination 2004 Irwin Rose Chemistry Mechanism of enzymatic ubiquitination 2000 Paul Greengard Med/Phys Signal transduction and brain protein kinases 1997 Paul Boyer Chemistry ATP synthase rotary catalysis mechanism 1997 John Walker Chemistry ATP synthase structure 1997 Jens Skou Chemistry Discovery of sodium, potassium ATPase 1994 Alfred Gilman Med/Phys Signal-transducing GTP-regulatory enzymes 1994 Martin Rodbell Med/Phys Signal-transducing GTP-regulatory enzymes 1993 Kary Mullis Chemistry Polymerase chain reaction 1992 Edmond Fischer Med/Phys Protein kinases 1992 Edwin Krebs Med/Phys Protein kinases 1989 Sidney Altman Chemistry Catalytic RNA 1988 Thomas Cech Chemistry Catalytic RNA 1988 Johann Deisenhofer Chemistry Structure of a photosynthetic reaction center 1988 Robert Huber Chemistry Structure of a photosynthetic reaction center 1988 Hartmut Michel Chemistry Structure of a photosynthetic reaction center 1982 Sune Bergstro¨m Med/Phys Prostaglandin biosynthesis 1982 Bengt Samuelsson Med/Phys Prostaglandin biosynthesis 1978 Peter Mitchell Chemistry Chemiosmotic principle 1978 Werner Arber Med/Phys Discovery of restriction enzymes 1978 Daniel Nathans Med/Phys Discovery of restriction enzymes 1978 Hamilton Smith Med/Phys Discovery of restriction enzymes 1975 John Cornforth Med/Phys Stereochemistry of enzyme-catalyzed reaction 1972 Christian Anfinsen Chemistry RNase folding 1972 Stanford Moore Chemistry RNase sequence and active-site chemistry 1972 William Stein Chemistry RNase sequence and active-site chemistry 1971 Earl Sutherland Med/Phys Discovery of 39,5-cyclic-AMP 1970 Louis Leloir Chemistry Structure and biosynthesis of sugar nucleotides 1970 Julius Axelrod Med/Phys Enzymatic synthesis of epinephrine 1964 Konrad Bloch Med/Phys Cholesterol metabolism 1964 Feodor Lynen Med/Phys Fatty acid metabolism 1961 Melvin Calvin Chemistry Photosynthesis 1959 Arthur Kornberg Med/Phys Enzymatic synthesis of DNA 1959 Severo Ochoa Med/Phys Enzymatic synthesis of RNA 1955 Hugo Theorell Med/Phys Mechanisms of redox enzymes 1953 Hans Krebs Med/Phys Citric acid pathway 1953 Fritz Lipmann Med/Phys Coenzyme A and fatty acid enzymology 1947 Carl Cori Med/Phys Enzymatic synthesis of glycogen 1947 Gerty Cori Med/Phys Enzymatic synthesis of glycogen 1947 George Wald Med/Phys Retinal cis-trans isomerization in visual processes 1946 James Sumner Chemistry Urease crystallization 1946 John Northrop Chemistry Protease crystallization 1937 Albert Szent-Gyo¨rgyi Med/Phys Vitamin C and catalysis of fumaric acid 1931 Otto Warburg Med/Phys Mode of action of respiratory enzymes 1929 Arthur Harden Chemistry Sugar fermentation pathway 1929 Hans von Euler-Chelpin Chemistry Fermentative enzymes 1922 Otto Meyerhof Med/Phys O2 and lactic acid metabolism 1907 Eduard Buchner Chemistry Cell-free enzyme-catalyzed reactions
aAlthough receptor-mediated endocytosis and prions have little to do with enzymes, their respective discoverers, Michael Brown (Nobel Laureate in Medicine and Physiology, 1987) and Stanley Prusiner (Nobel Laureate in Medicine and Physiology, 1997), both received their post-doctoral research training in enzymology under the late Earl R. Stadtman. 18 Enzyme Kinetics kinetics; Bisubstrate enzyme kinetics; Thermodynamics of hysteresis; Development of KINSIM and FITSIM software ATP hydrolysis of biochemical reactions; Application of for simulating enzyme rate processes; Herbert Fromm – Use Legendre transforms in biochemical thermodynamics; of reversible inhibitors (including product inhibitors, Christian Anfinsen, Stanford Moore and William Stein – alternative substrate inhibitors, as well as competitive Ribonuclease structure and folding, identification of cata- inhibitors) to distinguish multi-substrate kinetic mechanisms; lytic residues; John Albery and Jeremy Knowles – Novel Implications of abortive complex formation in enzyme isotopic approaches for defining the energetics of the triose- kinetics; Definition of kinetic reaction mechanisms (with phosphate isomerase and proline racemase reactions; Boyer) through isotope exchange measurements at thermo- Enzyme evolution, Catalytic efficiency, and Catalytic dynamic equilibrium; Constant-ratio approaches for perfection; Max Bergmann and Joseph Fruton – Poly-site analyzing three-substrate enzyme kinetics; Fallacy of ade- binding theory of enzyme specificity; Introduction of nylate energy charge hypothesis for ATP-utilizing/regener- synthetic N-carbobenzoxy-peptides as alternative substrates ating enzymes; Quentin Gibson – Development of stopped- for proteases and peptidases; Paul Boyer – Multi-substrate flow rapid mixing instrumentation; Heme-protein kinetics; enzyme kinetics; Definition of kinetic reaction mechanisms Herbert Gutfreund – Fast reaction kinetics of enzyme through the novel application of isotope exchange reactions; Kinetic criteria (with P. Boon Chock) for evalu- measurements at thermodynamic equilibrium; Oxygen-18 ating substrate channeling; Gordon Hammes – Temperature- tracer methods in carboxyl- and phosphoryl-group transfer jump reaction techniques to enzyme systems; Fast reaction reactions; ‘‘Binding-Change Mechanism’’ for rotary catal- kinetics of complex multi-enzyme processes; Brian ysis of ATP synthase (see Table 1.4: Nobel Laureates); Hartley – Chymotrypsin catalysis; Enzyme burst method for Britton Chance – Invention of the stopped-flow technique; detecting enzyme-bound, covalent reaction intermediates; First spectral detection of enzyme reaction intermediates; Charles Huang – Multisubstrate enzyme kinetics; Models First application of computers to simulate enzyme reaction for calcium ion complexation in calmodulin mediated acti- kinetics; Development of the Theorell-Chance bisubstrate vation of target enzymes; Kinetic analysis of allosteric kinetic mechanism; W. Wallace Cleland – Systematic enzymes; William Jencks – Catalytic strategies in chemistry enzyme nomenclature of multi-substrate enzyme kinetics; and enzymology; Conceptual basis for catalytic antibodies, Steady-state treatment of isotope exchange kinetics; Energetics and mechanism of calcium ion pump; Kaspar Development of exchange-inert metal-nucleotide com- Kirschner – Fast reaction kinetics of allosteric enzymes; plexes; Development of equilibrium perturbation technique Daniel Koshland – Induced-fit hypothesis; Sequential model to evaluate kinetic isotope effects for detecting rate-limiting for cooperativity of allosteric enzymes; Role of orbital chemical steps; Mildred Cohn and Albert Mildvan – alignment (Orbital Steering) in enzyme catalysis; Keith Oxygen-18 probes of P–O and C–O bond cleavage in Laidler – Application of absolute rate theory to enzyme phosphotransfer reactions; Development of NMR-based systems; Temperature and immobilization effects on distance measurements using proton relaxation in para- enzyme kinetics; Richard Lerner and Peter Schultz – magnetic environments; NMR approaches for defining Development of catalytic antibodies, based on a prediction enzyme exchange kinetics; Keith Dalziel – Development of by W. P. Jencks; Vincent Massey – pH Kinetics of fumarase; the F-parameter method for discriminating the order of Kinetic and mechanistic approaches in flavoenzyme catal- substrate binding by bisubstrate enzymes; Edward Dennis, ysis; Peter Mitchell – Chemiosmotic principle of trans- Pierre Desnuelle, Michael Gelb, Mahendra Jain and Robert membrane gradients (see Table 1.4: Nobel Laureates); Verger – Use of nonionic detergents and Langmuir troughs Jacques Monod, Pierre Changeaux and Jeffries Wyman – to investigate interfacial catalysis by lipases and phospho- Concerted transition model for allosteric interactions and lipases; Lipase processivity; Zacharias Dische – Discovery cooperativity; Dexter Northrop – Two-site ping-pong of allosteric feedback inhibition; Pierre Douzou and kinetics; Exploiting the Swain-Schaad relationship to isolate Anthony Fink – Development of ultra-low temperature and evaluate intrinsic kinetic isotope effects; Dieter Palm, (cryoenzymology) techniques to investigate enzyme kinetic Bryce Plapp and Judith Klinman – Kinetic isotope effects in properties; Manfred Eigen – Chemical relaxation process; enzyme-catalyzed hydride transfer; Role of quantum Temperature-jump technique; Prion protein polymerization mechanical tunneling in hydride transfer; Arthur Pardee and (see Table 1.4: Nobel Laureates); Fritz Eckstein, Jeremy Edwin Umbarger – Kinetics and feedback inhibition of Knowles, David Usher and Martin Webb – Stereochemical allosteric enzymes; Ephraim Racker – First demonstration probes of phosphomonoester- and phosphodiester-utilizing that covalent enzyme-substrate compounds are formed reactions; Alan Fersht – Site-directed mutagenesis as during enzyme catalysis; Michael Raftery – Early applica- mechanistic probes; Mechanisms for kinetic proofreading tion of secondary kinetic isotope effects to detect the oxa- by aminoacyl-tRNA synthetases; Novel approaches for carbenium ion intermediate formed in lysozyme catalysis; defining protein folding mechanisms; Carl Frieden –pH Irwin Rose – Isotopic probes of enol intermediates in kinetics of fumarase reaction; Three-substrate enzyme isomerases; Isotope trapping methods; Dynamic stereo- kinetics; Kinetic aspects of enzyme cooperativity and chemical probes (or positional isotope exchange); Chapter j 1 An Introduction to Enzyme Science 19
Elucidation of the ubiquitin ligase mechanism (see 3. Structural Mechanism – An atomic-level model Table 1.4: Nobel Laureates); Vern Schramm – Application of showing the structural basis for catalytic facilitation of multiple kinetic isotope effect data to the rationale design of the chemistry of substrate-to-product interconversion transition-state analogues for uses as specific, high-affinity as well as the physics of substrate adsorption and enzyme-targeted drugs; Earl Stadtman – Kinetic and regu- product release. latory behavior of signal transduction cascades via post 4. Regulatory Mechanism – A scheme offering a detailed translational modification, as demonstrated in his pioneering understanding of activator and inhibitor effects that are studies of enzyme-catalyzed adenylylation/deadenylylation a direct consequence of binding cooperativity, allosteric of Escherichia coli glutamine synthetase; Edwin Taylor, interactions with activators and/or inhibitors, post-trans- David Trentham, Clive Bagshaw and Martin Webb – lational modification, etc. Mechanoenzyme kinetics of actomyosin, as probed by fast Undertaking such investigations begins with elucidation of reaction kinetics, ‘‘photo-caged’’ ATP, and continuous a chemical reaction mechanism explaining all of the bond- assay with fluorescent phosphate-binding protein; Hugo breaking and bond-making steps needed to transform Theorell – Bisubstrate reaction kinetics of redox enzymes substrate(s) into product(s), as well as all detectable (see Table 1.4: Nobel Laureates); Frank Westheimer – elementary reactions comprising the kinetic scheme of Stereochemistry NADH hydride transfer; Stereochemistry enzyme interactions with substrates, intermediates, and of phosphoryl transfer, including pseudorotation; Kinetic products. Although many studies are initiated with the isotope effects; Photoaffinity labeling of enzyme active convenient use of unnatural substrates that are chromo- sites; Bioinorganic reaction mechanisms; Richard Wolf- genic or fluorogenic (i.e., the products of these weakly enden – Development of a rational basis for analyzing absorbing or fluorescing substrates have quantifiable transition-state inhibitor potency; Catalytic proficiency; and absorbance or fluorescence spectra), these studies should Jeffrey Wong – Theoretical treatment of steady-state enzyme ideally be carried out with the natural substrates to fully kinetics; Alternative substrate kinetics. understand the biological role of the enzyme under inves- Finally, those familiar with enzyme kinetics know that tigation. (In fact, altered reactivity of alternative substrates the complexity of certain enzymes generated such must always be anticipated.) The chemical and kinetic compelling interest that some enzyme chemists made mechanisms must be consistent with the reaction’s overall career-long commitments to the study of a single enzyme or stoichiometry, its stereochemistry, its kinetic and thermo- pathway. So strong was their attachment to their favorite dynamic properties, the location and energetics of rate enzyme that the late Ephraim Racker once told the author determining step(s), the structures of detected intermedi- that he was convinced that the perceived importance of an ates as well as any inferred transition state(s), as well as enzyme was often a manifestation of the interesting effects of temperature, pH, ionic strength, and solvent. The personalities investigating that enzyme. He was particularly structural mechanism begins with high-resolution struc- fond of the humanistic saying that ‘‘Interesting people make tures of the free enzyme as well as it complexes with for interesting enzymes.’’ In this respect, the above list is reaction substrate(s) and product(s), as well as any acti- admittedly incomplete and fails to acknowledge the vators or inhibitors of interest. But a structural interpreta- immense contributions of so many other creative and tion is incomplete unless it unifies the chemical, kinetic, interesting scientists. and regulatory mechanisms. The regulatory mechanism should explain how an effector molecule lowers (activa- 1.4 THE CONCEPT OF A REACTION tion) or raises (inhibition) the activation energy of one or more steps in the catalytic reaction cycle. Likewise, the MECHANISM effect of any post-translational modifications should be The chief ambition of enzyme chemists is to obtain the most reconciled with changes in the catalytic reaction complete description possible of an enzyme-catalyzed mechanism. reaction. An enzyme’s overall catalytic mechanism may be The optimal approach for integrating such information subdivided into four parts: is to construct rival hypotheses that make testable predictions connecting structure, energetics, and kinetics. 1. Chemical Mechanism – A reaction scheme showing all Ideally, these rival explanations will result in kinetically bond-breaking/-making steps, rearrangements, transi- distinguishable properties. Enzyme chemists make tion state(s), as well as the stereochemistry of partial strenuous demands on structural and chemical informa- and overall reactions. tion, and kinetic data often offer additional constraints for 2. Kinetic Mechanism – A scheme accounting for the deciding on the most likely of rival reaction mechanisms. time-dependent accumulation and breakdown of each Modern enzymology has benefited enormously from the enzyme-bound species, including the energetics of any atomic-level molecular structures, as provided by X-ray rate-determining step(s). crystallography and high-resolution, multidimensional 20 Enzyme Kinetics
NMR spectroscopy. Even so, while structural biologists enzyme cleaves peptide bonds within peptides and have glimpsed various stages of catalysis, there is no such proteins, acting preferentially at sites where the carboxyl- thing as a tell-all ‘‘motion picture’’ of even the simplest donating amino acid residue has a hydrophobic side-chain. catalytic process. These days, there are those enzyme The reaction is facilitated by push–pull proton transfer chemists who won’t believe anything without first seeing involving specific imidazole, carboxyl, and hydroxyl it, while others don’t see anything without first believing groups that are common to hundreds of other mechanisti- it. While we would desire to view catalysis from cally related enzymes in the ‘‘serine’’-protease super- a vantage point of quantum mechanics, the chief obstacle family. An acyl-serine intermediate permits one product to applying quantum mechanical approaches is that (designated by the R-group in Fig. 1.3) to dissociate, such enzymes are complex structures, frequently possessing that water can replace the departing amino group in 10,000–15,000 atoms, the positions of which are rarely a manner that leads to hydrolysis of the peptidyl acyl- known with adequate accuracy. Enzyme structures are enzyme and subsequent release of the second peptide also strongly influenced by seemingly countless non- fragment (designated by I9). Enzyme chemists are covalent bonding interactions, and each non-covalent reasonably confident of the general outline of the steps interaction contributes a relatively small increment to the illustrated in Fig. 1.3, especially in the light of the wealth conformational energy associated with an enzyme’s of structural, chemical, and kinetic information gleaned catalytically active conformation. Dealing with so many from persistent and systematic investigation. weak interactions remains a daunting challenge for Figure 1.4 illustrates the following key points about computer software developed to treat far simpler mole- serine-protease (and serine esterase) catalysis: (a) the cules. Even when quantum mechanical calculations are substrate and enzyme are structurally complementary with limited to a small segment or region within an enzyme respect to each other, with specificity determined by the (say the active site region), quantum mechanical and nature of charged residues deep within the active site; molecular mechanical models can quickly become (b) the mechanism exploits general base catalysis (see unwieldy. Even so, one can safely predict that, with Section 7.3.9: Brønsted Theory of Acid and Base Catal- advances in computer-based calculations, quantum ysis) by imidazole to activate the hydroxyl group of the mechanics may eventually prevail, as it promises to offer active-site serine residue; (c) the latter exhibits nucleo- the ultimate picture of catalysis. philic catalysis, as evidenced by the formation of a tetra- An enzyme mechanism must provide much more than hedral adduct; (d) the enzyme stabilizes the tetrahedral just the changes in covalent structure. A mechanism must transition state (and the transient covalent intermediate) also explain the enzyme’s actions during catalysis – all through hydrogen bonding between enzyme and interme- substrate binding interactions, all stereochemical trans- diates, particularly within the oxy-anion hole and by the formations, all pathways for product release, solvation electrostatic environment, provided in part by Asp-102; changes in active site, etc. The same also goes for changes (e) the reaction proceeds onward by means of general acid in coenzymes, cofactors and metal centers. Enzyme kinet- catalysis that facilitates the departure of the leaving group icists also seek to understand those structural, dynamic, and to form the acyl-enzyme (covalent) intermediate and catalytic changes that are the basis of an enzyme’s regula- departure of the amine (or alcohol) leaving group; and (f) tory behavior. Allosteric activators and inhibitors of the remaining steps in the catalytic cycle are formally the enzymes have the effect of respectively lowering and raising reverse of the above steps, resulting in hydrolysis of the reaction barrier(s), as do the activating and inhibitory effects acyl-enzyme, which commences with the imidazole group of post-translational covalent modifications of enzymes. activating water by general base catalysis, so as to facil- Allosteric transitions often involve a manifold of protein itate nucleophilic attack by water at the carbonyl carbon conformational states, the complexity of which imposes atom. A major limitation relates to an almost exclusive such kinetic ambiguity that one cannot reach penetrating reliance on synthetic chromogenic substrates (i.e., those conclusions about how an allosteric modifier alters generating a change in the substrate’s or product’s UV/ catalysis. visible spectrum upon peptide bond cleavage). Virtually nothing is known about the details (e.g., steady-state and fast kinetics, reaction cycle energetics, hydrogen bonding 1.4.1 Chymotrypsin: The Prototypical of the water substrate, conformational dynamics, as well as the formation and turnover of key intermediates) Biological Catalyst describing chymotrypsin catalysis when proteins serve as Chymotrypsin was among the earliest crystallized substrates. In this respect, the mechanism shown in enzymes, and its purity and abundance stimulated great Fig. 1.4 is still somewhat incomplete. interest in this amidohydrolase. The probable catalytic It is also worth emphasizing that despite the many steps mechanism for chymotrypsin has been worked out during in the catalytic reaction cycle, chymotrypsin is a powerful the past half-century of intensive investigation. This catalyst, as evidenced by the infinitesimally low Chapter j 1 An Introduction to Enzyme Science 21
FIGURE 1.3 Likely mechanism for chymotrypsin catalysis. Form-1 is the substrate-free enzyme, with its catalytic triad consisting of the solvent-inaccessible, side-chain carboxyl group of Aspartate-102, the side-chain imidazole group of Histidine-57, and the side-chain hydroxyl group of Serine-195. The location of these functional groups within the active-site cleft is depicted in the accompanying chymotrypsin structure (inset on upper right), based on the X-ray crystallographic work of David Blow. After substrate binding to an initial, reversible enzyme-substrate Michaelis complex, the catalytic triad in Form-2 facilitates nucleophilic attack by activating the otherwise poorly reactive serine hydroxyl group. A key point is that partial bond formation, and the resulting hydroxyl group polarization is sufficient to accelerate catalysis; formal ionization of the serine hydroxyl group is unlikely, because the alkoxide (pK near 15) is a far stronger base than the imidazole (pK¼6). Upon nucleophilic attack, the carbonyl group is converted to the tetrahedral ‘‘oxy-anion’’ intermediate (Form-3), a transition-state that is stabilized by two hydrogen bonds (dashed lines) supplied by two backbone peptide N–H groups from Glycine-193 and Serine-195. The oxy-anion spontaneously rearranges to form the covalent, acylated enzyme (Form-4). After the amine-containing product departs, the reaction cycle then proceeds with its second phase, commencing with the entry of water molecule into the active site (Form-5). Nucleophilic attack by this water molecule results in the second tetrahedral intermediate (Form-6), again stabilized by the hydrogen bond network. This second oxy-anion species spontaneously rearranges to form the reversible Michaelis complex (Form-7), with the active site occupied by the carboxyl group-containing product. The same double-displacement, or Ping Pong, pathway is likely to apply to hundreds of other members of the ‘‘serine’’ protease superfamily, including trypsin, elastin, and thrombin. Specificity is achieved by inter- actions with other substrate-recognition residues not indicated here.
uncatalyzed rate (k ¼ ~10 13 s 1 at pH 7 at 298 K) of enzyme. Enzyme chemists also learned for certain that peptide bond hydrolysis compared to the corresponding enzymes exploit a myriad of intermediates to achieve such reaction carried out in the presence of chymotrypsin (kcat ¼ high catalytic rate enhancements. ~10 s 1). The catalytic rate enhancement for chymotrypsin Finally, chymotrypsin is first biosynthesized as the is thus an astonishing 100,000,000,000,000! As discussed inactive storage form chymotrypsinogen. The latter is an throughout this textbook, the occurrence of covalent reac- example of a zymogen – an inactive enzyme precursor, from tion intermediates during catalysis in no way impedes an which an active enzyme can be generated enzyme-catalyzed 22 Enzyme Kinetics proteolysis. While still elongating from the ribosome, the placed such ideas on a firm footing. Even so, Nobel nascent polypeptide chain is directed to and translocated into Laureates Thomas Cech and Sidney Altman demonstrated the lumen of secretory granules, where it is oxidatively that certain RNA molecules are highly efficient catalysts processed to introduce essential –S–S– bonds. Upon for RNA self-splicing, phosphotransfer, and even hormone-stimulated release into the small intestine, peptide bond formation (Altman, 1993; Cech, 1993). These chymotrypsinogen is then cleaved between residues 15 and catalytic RNA molecules, also known as ribozymes, 16 by trypsin to yield two polypeptide chains that remain often achieve rate enhancements approaching 1011. The linked by means of a single disulfide bond. This peptide hammerhead-shaped ribozyme (Fig. 1.5) was the first RNA cleavage process (Reaction: Chymotrypsinogen þ H2O # motif observed to catalyze sequence-specific self-cleavage p-Chymotrypsin) generates an intermediate species, known by a magnesium ion-dependent transesterification. Con- as p-chymotrypsin, which has an imperfectly formed active taining only around 30 nucleotides in their catalytic cores, site and is hence a feeble catalyst. p-Chymotrypsin then these ribozymes are the smallest of the catalytic RNA undergoes autocatalysis (see Section 3.8.4), with peptide molecules. These enzymes display Michaelis-Menten bond cleavage (Reaction: p-Chymotrypsin þ H2O # kinetics in their action on substrates (see Section 5.6: Chymotrypsin þ Peptides) achieved through the action of Ribozyme Kinetics), with Michaelis constants (Km) values chymotrypsin and itself forms a second fully active chymo- ranging from 20 to 200 nM and turnover numbers (i.e., 1 trypsin molecule. The latter consists of three disulfide-linked kcat) in the range of 0.03 s . Product release is generally polypeptides: Chain-A, the N-terminal region ending at fast, suggesting that the rate-determining step is phospho- residues 1 to 14; Chain-B, the longest chain comprising diester bond-scission. residues 16 to 146; and Chain-C, comprising the C-terminal Ribozyme-mediated phosphoryl transfer appears to region, beginning at residue 149. Note that two short involve destabilization of the substrate’s ground-state peptides, consisting of residues 14–15 and 147–148, have no (see also Section 1.5.4: ‘‘Reacting Group Approxima- catalytic role and are released to form the active enzyme. tion, Orientation and Orbital Steering’’ under Section Fully active chymotrypsin possesses an ‘‘oxy-anion hole’’ 1.5: Explaining the Efficiency of Enzyme Catalysis). that accommodates the negatively charged tetrahedral Magnesium ion complexation and hydrogen bonding intermediates already described in Fig. 1.3, thereby affording stabilize the negative charge that develops on the yet another way to promote catalysis by stabilizing an leaving group during entry of the nucleophile. This obligatory reaction intermediate. transesterification reaction is mechanistically analogous to that used in the mRNA spliceosome as well as in 1.4.2 Ribozymes other DNA topoisomerase and transposition reactions. The true catalytic nature of the ribozyme was demon- From the earliest times, enzymes were always associated strated by the discovery that the RNA component of with proteins, and the inspired work of Nobel Laureate RNase P catalytically processed tRNA precursors John B. Sumner on the crystallization of jack bean urease (Altman, 1993).
Catalytic NH Loop-3 2 RNA , N N 5'-End , N Stem-3N N, O N N , N A U O P O O , , A X , , , , O 5 3 N N , , , , A CleavageN N HO N N N N G Site Loop-1 Loop-2 N N N N 2+ , O Me (OH 2) 4 N N N N C Stem-1 Stem-2 A O G U P O N A O O FIGURE 1.4 Generalized base-pair structure of hammerhead ribo- 5'-Leaving zymes. Shown are consensus nucleotide residues (marked G, C, A, U) Group within the central ring consisting of 17 nucleotides (aqua) as well as vari- able nucleotides (N). This secondary structure is stabilized by three runs of hydrogen-bonded nucleotide pairs forming the same type of ‘‘stem-and- Scheme 1.4 loop’’ structural elements that are frequently observed in folded, single- stranded messenger RNA and ribosomal RNA. The central ring and the variable-length loops (indicated by dashed lines) facilitate folding into Scheme 1.4 illustrates the likely catalytic path for self- a compact, sphere-shaped tertiary structure. Self-cleavage site is indicated splicing reaction of group-II introns, which requires the in red. proper folding of intronic RNA into its enzymatically Chapter j 1 An Introduction to Enzyme Science 23 active form. The reaction mechanism probably Enzyme science has traditionally focused on the organic commences with metal ion-assisted loss of a proton ribose chemistry of biochemical reactions, particularly the changes 29-OH, allowing the incipient 29-alkoxide to attack the in covalent bonding as substrate is transformed into product. phosphodiester. Upon forming a pentavalent oxy- This rewarding enterprise helped to establish the role of phosphorane intermediate (with opposing nucleophile and countless covalent and ionic intermediates as well as the exiphile), the rate-limiting step is likely to involve P–O role of coenzymes and other cofactors. It’s a historic fact, bond scission. The active-site metal ion both facilitates however, that Boyer’s discovery of the ATP synthase intronic RNA folding as well as stabilizes the transition mechanism was delayed by the failure of researchers to state. With the notable exception that pancreatic Ribo- realize that the driving force for ATP synthesis was not nuclease A employs imidazole group in place of a metal a high-energy covalent intermediate, as ironically he had iontopolarizethe29-OH, the catalytic reaction cycles of himself originally proposed (Boyer, 2002). Peter Mitchell’s RNase and self-splicing reaction of group-II introns are chemiosmotic principle ultimately illuminated the need to remarkably similar. rationalize how Gibbs free energy, stored in the form of The discovery of catalytic RNA reminds us that one a transmembrane proton gradient, can drive ATP synthesis þ should not dismiss the possibility that other biological from ADP, Pi, and H , and vice versa. substances (e.g., polysaccharides, complex lipids, etc.) may Contemporary biochemistry has demonstrated time and prove to be biological catalysts. The phenomenon of time again that many reactions have: (a) substrate-like or micellar catalysis, for example, is already firmly rooted in product-like protein conformational states differing only in modern organic chemistry. In fact, some micellar catalysts their non-covalent bonding interactions: or (b) substrate- even exhibit chiral recognition (i.e., the capacity to combine like or product-like state corresponding to transmembrane with and transform substrate molecules in a stereoselective solute gradients. Various ATP- and GTP-dependent molec- manner). The likely role of biomembranes in catalysis ular motors, for example, rely on the free energy of ATP remains to be determined. hydrolysis to drive protein conformational changes, which in turn drive processes like muscle contraction, organelle 1.4.3 Mechanoenzymes trafficking, and cell crawling. Structural metabolism represents the ceaseless building-up and tearing-down of the Over the past century, biochemists have discovered cell’s macromolecular and supramolecular structure literally thousands of different enzyme catalyzed reac- through the ATP- and GTP-dependent affinity-modulated tions. A compilation by Purich and Allison (2002) puts interactions of chaperonins and proteasomes, molecular the number at nearly 7,000 unique catalytic activities, but motors, pumps, latches, and switches. Other reactions, such data from various genome projects suggest there are as the facilitated exchange of tightly bound protein–ligand likely to be another three to five thousand more enzymes complexes or membrane carriers, strictly involve changes in whose reactions remain to be defined. A large number non-covalent bonding and proceed without the breaking/ appear to be protein kinases, receptor-linked GTP-regu- making of even a single covalent bond. In short, mecha- latory proteins, and chromatin remodeling enzymes, as noenzyme catalysis involves non-covalent substrate-like well as enzymes mediating micro-nutrient metabolism. and product-like states, and the failure to include these in Based on the ways that enzymes break, rearrange, and describing mechanoenzyme reaction has led to confusion in form covalent bonds, and guided largely by organic enzyme nomenclature and classification. chemical principles that distinguish reaction types, the To provide a rational framework for the systematic Enzyme Commission defined the following classification classification of enzymes, including mechanoenzymes, scheme. Purich (2001) offered a new definition for an enzyme: Class-1: Oxidoreductases – catalyze oxidation/reduction ‘‘An enzyme is a biological catalyst for making and/or reactions. breaking chemical bonds.’’ Class-2: Transferases – catalyze group-transfer reactions. While appearing to be no more encompassing than existing definitions of enzyme catalysis, the crucial Class-3: Hydrolases – catalyze hydrolytic cleavage of difference lies in the use of chemical in place of covalent covalent bonds. to describe the bonding changes. This definition Class-4: Lyases – catalyze addition and elimination of func- acknowledges those enzymes catalyzing the interconver- tional groups to unsaturated and saturated carbon atoms. sion of non-covalent substrate- and product-like states or conditions: Class-5: Isomerases – catalyze rearrangement of atoms or groups of atoms. Interaction State-1 + ATP + H20 Interaction State-2 + ADP+ Pi Class-6: Ligases – catalyze joining of molecules or func- tional groups. Scheme 1.5 24 Enzyme Kinetics
Biological catalysis of this type is observed in instances nucleotide exchange, and this property is indistinguishable where the substrate is a protein with a very slowly disso- from the cardinal feature of all catalysts, namely transition- ciating ligand. An example is the adenine nucleotide state stabilization. Such considerations demonstrate unam- exchange reaction of the cytoskeletal protein actin. biguously that biological catalysis can take place without Hydrolysis of actin-bound ATP during cell motility leads to the breaking and making of covalent bonds. the formation of tightly bound Actin$ADP. Spontaneous In his timeless book The Nature of the Chemical Bond, exchange of solution-phase ATP with Actin$ADP to Linus Pauling (1945) offered the following definition that regenerate Actin$ATP is too slow to sustain the high fila- has guided my thinking about enzyme catalysis: ment assembly rates (400–500 monomer/filament/sec) needed to sustain cell motility. To overcome this kinetic ‘‘We shall say that there is a chemical bond between two obstacle, motile cells have high concentrations of profilin, atoms or groups of atoms in case the forces acting between a 15-kDa regulatory protein that catalyzes the following them are such as to lead to the formation of an aggregate with sufficient stability to make it convenient for the chemist protein–ligand exchange reaction: to consider it as an independent molecular species.’’
Profilin + Actin·ADP Profilin·Actin·ADP Significantly, Pauling made no mention of covalent bonds, stressing instead the unifying nature of chemical bonds. Profilin·Actin·ADP [Profilin·Actin·__ ]‡ + ADP That many protein conformational states and numerous protein–ligand complexes have been shown to be suffi- ‡ [Profilin·Actin·__ ] + ATP Profilin·Actin·ATP ciently long-lived to exhibit chemically definable properties suggests that transformations in these non-covalent inter- Profilin·Actin·ATP Profilin + Actin·ATP actions ought to be treated as chemical reactions. And with Scheme 1.6 modest tinkering, Pauling’s definition of a chemical bond can be extended to include the persistent, definable position Red- and blue-colored nucleotides are used in Scheme of a solute relative to the inner and outer faces of 1.6 to indicate that the reaction is one of physical exchange, a membrane. Solutein and Soluteout therefore represent as opposed to the transfer of a phosphoryl group from substrate-like and product-like states in reactions catalyzed unbound ATP to form actin-bound ATP. Profilin accelerates by passive transporters (e.g., Solutein # Soluteout) and this reaction by a factor of 150, and profilin’s action is active transporters (e.g., Solutein þ ATP # Soluteout þ without effect on the exchange reaction equilibrium. As ADP þ Pi; or, Solutein þ Gradient-State1 # Soluteout þ shown in Fig. 1.5, profilin binds preferentially to nucleotide- Gradient-State2). The now classical work by American free actin, approximately 12-times more tightly than to biochemist Ronald Kaback demonstrated how lactose actin$ATP, and 72-times more tightly than to actin$ATP permease couples lactose transport to a transmembrane (Selden et al., 1999). Profilin’s preferential interaction with proton gradient. nucleotide-free actin explains its ability to promote Another example of non-covalent catalysis is the Naþ- glucose symport system, which mechanochemically links the energy stored in a transmembrane sodium gradient to + X+ drive glucose uptake. This transporter operates by the same random substrate addition mechanism as that observed with Uncatalyzed enzymes like hexokinase and creative kinase.
+ + + Na Glc Na Glc G PE▪X
Catalyzed + Tout Na Tin Glc
Complex2 T T P + AD+T out + + in E Complex P +AT+ D Tout Glc Na Tin Glc Na 1 E + Tout Glc Tin Na
Reaction Progress Glc Na+ Glc Na+ FIGURE 1.5 Profilin catalysis of exchange of solution-phase ATP with actin-bound ADP to form solution-phase ADP with actin-bound Scheme 1.7 ATP. Symbols used are: A, Actin, AD, Actin$ADP ¼ Substrate; AT, Actin-ATP ¼ Product, PE, Profilin acting as an Enzyme; Complex1 ¼ In Scheme 1.7, the isomerization of the central pathway Profilin $Actin$ADP ¼ Enzyme$Substrate Complex; Complex ¼ E 2 represents the conversion of the transporting enzyme from ProfilinE$Actin$ATP ¼ Enzyme$Product Complex. Note: Profilin catalyzes physical exchange of the entire nucleotide molecule, and not its outside conformation Tout to its inside conformation Tin. phosphoryl transfer. Only when the sodium ion and glucose sites are occupied Chapter j 1 An Introduction to Enzyme Science 25 does the symporter operate. Note again that no covalent components are formed, remodeled, and degraded enzy- bond-making/-breaking steps are involved. Binding of matically. Endocytosis and organelle traffic, cell crawling, sodium ion actually increases the affinity of enzyme for signal transduction, and mitosis/meiosis are processes that glucose to such an extent that greatly favors the upper path are taking on the appearance of the pathways of intermediary (Crane and Dorando, 1980). metabolism. Even long-term potentiation, a neuronal Foldases are mechanoenzymes that catalyze rate- process lying at the root of our memory and consciousness, is limiting steps along the folding pathway of a protein, now known to depend on actin polymerization motors to including the cis-trans isomerization of peptidyl-prolyl maintain and/or remodel dendritic spines into synapses. bonds as well as the formation/isomerization of disulfide Because these energy-driven, affinity-modulated mechano- bonds. Molecular chaperones (sometimes regarded to be enzymes must be distinguished from energy-dissipating a specialized class of foldases) are highly conserved hydrolases (e.g., ‘‘ATPases’’ and ‘‘GTPases’’), Purich conformation-isomerizing enzymes found in all living (2001) indicated the need for an additional enzyme class: systems. They facilitate folding by interacting with mis- folded polypeptide chains, but they do NOT become part of Proposed New Class: Energases – catalyze the transduc- the final structure or alter the equilibrium poise of the tion of chemical-bond energy into noncovalent interactions that generate force and do work. Proteinunfolded # Proteinfolded equilibrium. Among the best- characterized molecular chaperones are GroEL-GroES and While instituting a new class represents a challenging DnaK-DnaJ-GrpE systems that are found in the cytoplasm task – one involving upwards of 1,000 enzymatic activities, of Escherichia coli. Other molecular chaperones include those resisting such change ignore the obvious: enzyme Clp ATPases, HtpG and IbpA-IbpB. names and classes should account for the entire chemical As will be discussed in Chapter 12, non-covalent reaction – and not just the covalent chemical bonds. substrate-like and product-like states are of paramount Finally, although many of the enzymes described here importance in the action of mechanochemical enzymes (or are relatively feeble catalysts (e.g., profilin’s rate enhance- simply mechanoenzymes). These highly specialized ment e is only ~140–150), especially compared to other enzymes use chemical bond energy to perform work (i.e., enhancement factors of 1015, the phenomenon of catalysis generate a force F over a distance Dx). Chemical-to- has nothing to do with the magnitude of rate enhancements. mechanical energy transduction is accomplished by means If the uncatalyzed reaction (or reference reaction) is already of an affinity-modulated binding interaction, generally fairly rapid, the catalytic rate enhancement need not be great using the Gibbs free energy of ATP (or GTP) hydrolysis to for the catalyzed rate to proceed on a physiologically control the strength of their binding to their metabolic target meaningful time-scale. Natural selection provides a ration- (e.g., other enzymes, proteins, transported substances, ale for the attainment and maintenance of evolutionary cytoskeletal and membrane components, as well as nucleic advantages. Mutations making an enzyme more efficient acids). Although each mechanoenzyme has its distinctive than necessary (i.e.,‘‘over-perfection’’) offer the cell no mechanistic features, the general scheme can be depicted as durable advantage, and may even prove to be deleterious follows: (e.g., by allowing undesirable accumulation of pathway intermediates). Simply put, an enzyme need only be as good a catalyst as Nature demands in the context of the overall StateS + Enz·ATP StateS·Enz-ATP biochemical process.
+ StateS·Enz·ATP + H20 StateS*·Enz·ADP·Pi·H 1.5 EXPLAINING THE EFFICIENCY + + StateS*·Enz·ADP·Pi·H StateP·Enz·ADP + Pi + H OF ENZYME CATALYSIS Biochemists and chemists alike have struggled to explain State ·Enz·ADP + ATP State + Enz·ATP + ADP P P why enzyme catalysis is so extraordinarily fast. As stated by + Warshel et al. (2006), ‘‘the real puzzle is why the enzyme StateS + ATP + H20 StateP + ADP + Pi + H reaction with the specific chemical groups (e.g., acids and Scheme 1.8 bases) is so much faster than the reaction with the same groups in solution.’’ The efficiency of biological catalysis is in fact so great where the braces are used to indicate complexes, and the that the best way to assess the efficiency is to compare the asterisk indicates a conformationally energized species. free energies of activation DGact, which are by definition Note also the various states where the mechanical work can proportional to ln(k). When comparing catalyzed and be accomplished. The field of cell biology can be largely uncatalyzed processes, it is also essential to compare the regarded as structural metabolism, where the supramolecular activation energies for and enzyme-catalyzed reaction and 26 Enzyme Kinetics
50 some point in their respective catalytic cycles, they remain ΔG+ cat tenaciously associated with their reaction intermediates ΔG+ 40 w,w until catalysis is accomplished. Their active sites are also ΔG+ p,w highly flexible, facilely adapting to meet the needs for rapid 30 acid-base and/or electron transfer reactions. When combined with their substrate, these active sites serve as
20 ideal ‘‘solvents’’ – at times aqueous protic solvents, and at (kcal/mol)
G G other times nonaqueous protic solvents, while always 10 stabilizing the succession of enzyme-bound intermediates comprising a catalytic cycle. 0 As we shall see throughout this book, virtually all classes 1 2345 67 8910 11 12 13 14 15 16 17 18 of organic reactions observed in the chemical laboratory Reaction System have one or more enzyme counterparts. Much as the most FIGURE 1.6 Activation free energies for representative enzymatic successful chemists, who exploit the laboratory to improve reactions (DGcat), reference reactions operating by the same mecha- the rates and yields of these reactions, enzymes have nism (DGp,w), as well as the actual mechanism in water (DGw,w). The exploited evolution to become highly effective catalysts. At reactions are those catalyzed by: 1, ketosteroid isomerase; 2, aldose reduc- tase; 3, carbonic anhydrase; 4, chorismate mutase; 5, trypsin; 6, haloalkane this point, they have developed highly effective mechanisms delahogenase; 7, alkaline phosphatase; 8, Ras GTPase complexed to its that doubtlessly take fullest advantage of catalytic strategies activating protein GAP; 9, triose phosphate isomerase; 10, acetylcholine as described in Sections 1.5.1 through 1.5.11, but optimized esterase; 11, lysozyme; 12, RNase (mono-ionic intermediate); 13, RNase from start to finish for extreme efficiency. The late British- (di-ionic intermediate); 14, ATPase; 15, bacteriophage T7 DNA poly- American chemist Jeremy Knowles adopted the title merase; 16, orotidine 59-monophosphate decarboxylase; 17, exonuclease activity of DNA polymerase I (Klenow fragment); and 18, staphylococcal ‘‘Enzyme Catalysis: Not Different, Just Better’’ for his nuclease. Figure and legend reproduced from Warshel et al. (2006) with cogent discussions of catalytic rate enhancement (Knowles, permission of the authors and publisher. 1991). His view was that enzymes operate by highly per- fected catalytic mechanisms that, with the exception of their the corresponding reaction (i.e., the reference reaction) that speed and specificity, resemble those explored for decades operate by the very same mechanism, rather than just the by physical organic chemists. That said, the development of same chemical reaction as it occurs in water. Figure 1.6 a precise quantitative model for enzymatic rate enhance- presents such a graph from Warshel et al. (2006) comparing ment, even for a single enzyme, remains an elusive goal. eighteen different reactions in terms of DGcat, the activation While various explanations discussed below are based on energy for the enzymatic reactions, DGp,w, the activation principles from physical organic chemistry, nearly all focus energy for the corresponding reference reactions operating on the stability of enzyme transition states and/or the by the very same mechanism in water, as well as DGw,w, the dynamic flexibility of enzymes. activation energy for the actual mechanism in water. Among the explanations for such phenomenal efficiency are: the use of binding energy to stabilize reaction transition 1.5.1 Stabilization of Reaction Transition states, the catalysis-promoting role of electrostatics in States stabilizing transition states, the role of reactant approxi- Without specifying how, J. B. S. Haldane (1930) offered mation and orientation (including orbital steering) in the idea that enzymes lower the activation energy for guiding substrate interactions with catalytic groups, the role catalysis. Linus Pauling (1946; 1947) took the matter of low-barrier hydrogen bonds in stabilizing reaction tran- further by attributing enhanced catalysis to an enzyme’s sition states, the coordination of acidic and basic groups in ‘‘pushing’’ or ‘‘pulling’’ protons to and from reactants, the ability to interact with and stabilize the reaction transition role of the enzyme in destabilizing substrate ground states, state (a proposal now known as Transition-State Stabilization). the formation of covalent intermediates in preserving group transfer potential, the roles of metal ions as templates, + Lewis acids, and special redox states, as well as the catalytic EX+ + role of enzyme conformational dynamics, including Stabilize EX+ + inherent force-sensing, force-managing and force-gener- EX+ ating mechanisms. E+S E+S While enzymes simply must decrease the activation energy for the reactions they catalyze, determining exactly ES ES how this is accomplished has stubbornly resisted quantita- tive explanations. Part of the answer is that enzymes are The idea was that each enzyme becomes structurally most often catalytically processive, meaning that, beyond complementary to the transition state, such that the Chapter j 1 An Introduction to Enzyme Science 27 geometry, polarity, and electrostatic charge of the enzyme stabilize ionic and polarized transitions states. The presence and the transition-state configuration of the substrate are of such charged groups actually makes the active site’s local mutually stabilizing. Pauling (1947) wrote: environment significantly more polar than water (Warshel and Floria´n, 1998), allowing ionic transition states to be From the standpoint of molecular structure and the stabilized by nearby fixed dipoles. The nucleophilic and quantum mechanical theory of chemical reaction, the electrophilic properties of functional groups on the catalyst only reasonable picture of catalytic activity of enzymes and reactant are also increased by dehydration of the cata- is that which involves an active form of the surface of lytic center. In addition, electrostatic attraction and charge the enzyme which is precisely complementary in structure not to the substrate molecule itself, but rather to the neutralization tend to release water from enzyme active substrate molecule in a strained configuration correspond- sites, thereby exerting a powerful activating effect on ing to the ‘activated complex’ for the reaction catalyzed by nucleophilic reactions. the enzyme: the substrate molecule is bound to enzyme, Note that stabilization of the very same transition state in and caused by forces of attraction to assume the strained bulk water would require a substantial thermodynamic state which favors the chemical reaction – that is, the acti- penalty, referred to as a reorganization energy, for water vation energy of the reaction is decreased by the enzyme to molecules to be arranged in a manner that stabilizes ionic an extent as to cause the reaction to proceed at an appre- transition states. In enzymes, the ionic groups are pre- ciably greater rate than it would in the absence of the organized by protein folding, such that the resulting facili- enzyme. tated catalysis is attended by a very small reorganization The key point is that the enzyme need not initially be energy. Folding of the enzyme creates a constellation of complementary to the transition state configuration. An positively and negatively charged functional groups that are active site that accommodates a reaction transition state appropriately positioned for optimal catalysis. This concept would also tend to stabilize those forms of the substrate that may be extended to include catalysis-promoting changes in most closely resemble the transition state both geometri- electrostatic interactions as reactants proceed through the cally and/or electronically. Transition-state stabilization reaction cycle, including effects of conformational changes thus makes it easier for the substrate to reach and surmount and hence enzyme dynamics on electrostatic interactions the transition-state, and the net effect should be greatly and vice versa. enhanced chemical reactivity. Coulombic interactions mainly occur among acid and Note that little advantage would be gained if an enzyme base groups in the enzyme (as well as ionizable groups were to stabilize both the E$S and E$Xz equally, because the with its substrate). Metal ions also play important roles in activation energy would remain the same as that occurring electrostatic catalysis. In some cases, other permanently in the absence of the enzyme. charged side-chain groups (e.g., the guanidinium of argi- nine and the quaternary ammonium group of 3-methyl- + + EX+ EX+ histidine) may contribute to electrostatic stabilization of + + Stabilize EX+ EX+ transition states. Another advantage of electrostatic effects + EX+Only Stabilize Stabilize + is that they are ‘‘tunable,’’ meaning that the local envi- ES Only ES & EX+ E+S E+S E+S E+S ronment can alter the pKa values of acidic and basic groups. For example, when placed into a hydrophobic ES ES environment, acids tend to exhibit higher pKa values (i.e., ES ES formation of the carboxylate anion –COO is disfavored), whereas bases tend to have lower pK values (i.e., As we shall see in Section 8.6, these ideas are also a formation of cationic –NHþ groups is disfavored). When consistent with the action of both naturally occurring and 3 in the vicinity of a residue of like charge, acids likewise synthetic enzyme inhibitors that are structurally analogous tend to exhibit higher pK values, whereas bases tend to to the reactant’s transition-state. By mimicking the transi- a have lower pK values. When in the vicinity of a residue of tion state, these analogues can bind to an enzyme with a opposite charge, acids likewise tend to exhibit lower pK extraordinary affinity, simply because the enzyme need not a values, whereas bases tend to have higher pK values. divert a great deal of its binding energy to rearrange the a Finally, a-helices also have associated dipole moments analogue into a configuration resembling the reactant in its that can also exert electrostatic effects on active-site transition state (Schramm, 2003; Wolfenden, 1969). functional groups (Hol, 1985). Knowles (1991) discussed how one particularly well-aimed helix in triose phosphate 1.5.2 Electrostatic Stabilization isomerase is trained on His-95, lowering the pKa value of the latter from an unperturbed value of 6 to below 4.5. of Transition States Charge neutralization can also exert a strong desolvating As the name implies, electrostatic catalysis is the conse- effect within active sites. Prior to neutralization, each quence of the strong local Coulombic interactions that active-site cation and anion binds several water molecules, 28 Enzyme Kinetics
xþ y such that [Cation$(H2O)k] þ [Cation$(H2O)l] ¼ of hydrogen bonding in fostering catalysis. Two hydrogen [Salt$(H2O)(kþl) m] þ mH2O. bonds stabilizing the oxyanionic tetrahedral intermediate in Warshel et al. (2006) presented persuasive arguments chymotrypsin catalysis can contribute 7–8 kcal/mol of that the catalytic power of enzymes may well be almost transition-state stabilization, resulting in considerable entirely the consequence of electrostatic stabilization of catalytic rate enhancement. the transition state. Among the many examples, two classical cases are the lysozyme reaction, for which the 1.5.3. Intrinsic Binding Energy oxacarbenium ion is stabilized by nearby carboxyl groups, and the chymotrypsin reaction, in which the Binding energy effects arise from the sum total of favorable tetrahedral oxy-anion intermediate is stabilized by the non-covalent interactions between an enzyme and its intrinsically electrostatic phenomenon of hydrogen substrate(s), including a substantial contribution from van bonding. der Waals interactions associated with structural comple- mentarity of the enzyme and its substrate as well as desol- vation. The favorable enthalpy of substrate binding is O thought to overcome the unfavorable entropy associated OH O with bringing two (or more) molecules together. Once O O formed, the E$S complex allows the catalysis to be effec- O O tively an intramolecular process. HO Jencks (1975) suggested that enzymes gain great HO advantage over ordinary catalysts by managing the energy OH of binding interactions to orient substrates relative to each other and with respect to catalytic groups within the active The lysozyme mechanism was first analyzed computa- site. Page and Jencks (1971) showed that the loss in tionally by Warshel and Levitt (1976), who were among the entropy in going from a bimolecular to a unimolecular earliest proponents of computer-based molecular modeling reaction (i.e., E þ S # E$S) results in the loss of trans- to assess the origin of enzyme rate enhancements. Ideally, lational, rotational and vibrational degrees of freedom, thus one desires a quantum mechanical (QM) model defining all accounting for around 108 of the rate enhancement the atoms in the reactant and catalyst. An inherent limitation observed in enzyme-catalyzed reactions. Based on results on QM calculations, however, is that the required compu- of their computer modeling of subtilisin interactions with tational time rises very steeply with increasing numbers of model substrates, Villa´ et al. (2000) reached a completely atoms and electrons in the molecule(s) of interest, making different conclusion – namely that the contribution of DSz studies of entire enzyme-reactant interactions totally to DGz is much smaller than previously thought. They unworkable. To maneuver around this limitation, Warshel suggest that this is true because: (a) many of the motions and Levitt (1976) pioneered a combined QM and molecular that are free in the reactant state of the reference reaction mechanical (MM) approach, restricting the quantum are also free at the transition state; (b) the binding to the chemical description to the reaction center, while relying on enzyme does not completely freeze the motion of the a computationally efficient classical treatment for the reacting fragments, so that DSz in the enzyme is not zero; remainder of the molecule. Based on their QM/MM results and (c) the binding entropy is not necessarily equal to z with lysozyme, Warshel and Levitt (1976) suggested that DS water. the positive charge developing on the C-1 carbon of the glucopyranosyl residue would be stabilized by the adjacent, 1.5.4 Reacting Group Approximation, electron-rich ring oxygen and the charge-neutralizing effects of nearby glutamate and aspartate. Importantly, Sun, Orientation and Orbital Steering Liao and Remington (1989) used classical electrodynamics Substrate binding to the active site promotes catalysis: to find that C–O bond breakage and the consequent charge (a) by converting multi-substrate reactions from bimolec- separation is promoted by a large electrostatic field across ular rate processes to what essentially becomes a uni- lysozyme’s active-site cleft, created in part by a very molecular rate process; (b) by increasing the effective local asymmetric distribution of charged residues on the enzyme concentration of reactants with respect to each other; and (c) surface. That other lysozymes of unrelated primary by arranging and orienting reactant functional groups with sequence have similarly distributed charged residues and respect to each other. In most instances, intramolecular electric fields suggests the generality of electrostatic stabi- reactions occur at much faster rates than corresponding lization (~9 kcal/mol) as the basis for catalytic rate bimolecular reactions, and both proximity and orientation enhancement in lysozyme. can increase the effective local concentrations of reactants. Because the hydrogen bond is primarily an acid-base As discussed in Chapter 3, this behavior is related in part to neutralization, electrostatic catalysis also explains the role the brief lifetime of collision and encounter complexes, Chapter j 1 An Introduction to Enzyme Science 29 leaving fleetingly short times for productive chemical polarization, E$S conformational change, and/or electro- reactions to occur. Proper placement and orientation of static effect would presumably be required. reactive groups is also recognized to play a major role in catalytic rate enhancement through stereo-electronic assis- EX + tance, wherein reactants are arranged for maximal reac- Stabilize EX+ + tivity. Of course, orientation comes at a price (i.e., often EX+Only manifested by a decrease in entropy) which must be offset Form Unstable by some other favorable catalyst–substrate binding inter- ES Complex ES ES actions in regions that are immediately adjacent to the bonds E+S that are broken and/or made during catalysis. E+S Inspired by the then obvious success of the conservation- of-orbital-symmetry rules (Woodward and Hoffmann, Perhaps the best-known version of ground-state destabi- 1970) in explaining reactivity, Storm and Koshland (1970) lization is the Circe Effect. Jencks (1969) suggested that proposed and Dafforn and Koshland (1971; 1973) suggested highly favorable substrate binding interactions in a substrate’s that enzymes may promote catalysis by precisely aligning nonreactive region may facilitate catalysis by forcing the (steering) the molecular orbitals of their substrates. In the substrate’s reaction center into a destabilizing environment. earliest versions of Orbital Steering, enzyme-enforced The Circe Effect is viewed as using substrate binding energy constraints on molecular orbital alignment were viewed as to help reacting groups to approach the transition state. The so restrictive as to be physically unrealistic, and the name of this effect derives from the mythic Greek enchantress proposal was roundly dismissed on the basis of the antici- Circe whose sweet songs beguiled passersby to her island, pated high thermodynamic penalty for extremely precise where they were then transformed through the action of her orbital alignment and the weak dependence of force various spells and potions. constants on slight changes in bond angle (Bruice, 1972; Bruice (2002) suggested that an enzyme positions its Jencks and Page, 1974). substrate(s) in a conformation, such that thermal fluctua- Noting that the contribution of orbital steering to tions allow that conformation to easily surmount the barrier catalytic rate enhancement cannot be quantified in the to reaction. The basic idea is that for covalent bond absence of an accurate means for correlating structural formation, reacting atoms of substrate and enzyme must interactions and catalytic enhancement, Scott (2001) first come together within a suitable reaction distance (say argued that orbital steering may explain aspects of RNA 3–4 A˚ ) and approach angle (say 5–10 ), such that suitably catalysis. For ribozymes, orbital steering appears to be rearranged and highly reactive ground-states, termed Near- fortuitously uncoupled from conformational, distance and Attack-Conformers (NACs), would thereby accelerate orientation effects. During hammerhead ribozyme catal- catalysis. In this explanation, the enzyme might bind ysis, two conformational changes appear to align the strongly to a transition-state structure, but this binding orbitals of reacting atoms, and Scott (2001) suggested that energy is not thought to be released specifically to speed each of these two conformational changes is likely to the reaction (Luo and Bruice, 2004; Torres, Schiott and provide rate enhancement 3 of ~1,000. With an overall Bruice, 1999). Except for the speculative role of anti- rate enhancement of 106 that is solely attributable to correlated motions of proximal residues in destabilizing the orbital steering, Scott (2001) suggested that orbital steer- substrate (Luo and Bruice, 2004), the notion of NACs ing is a significant factor in the catalysis of ribozymes and merely restates the obvious, in that reactant-state destabi- protein enzymes. lization is merely an alternative description of transition- For additional comments on orbital steering and its state stabilization. Warshel et al. (2006) noted that, if both implications, the interested readers should consult valuable the reactant state (RS) and transition state (TS) for an reviews by Hackney (1990) and Mesecar, Stoddard and enzyme-catalyzed reaction were to have similar charge Koshland (1997). distributions, the same preorganization effects are apt to stabilize the RS and TS, leading to an apparent NAC effect by making the RS structure closer to that of the TS. They thus argue that the so-called NAC effect is an expected 1.5.5 Reactant State Destabilization result of the TS stabilization rather than the underlying In this case, the enzyme strains or distorts the substrate cause of catalysis. while still in its ground-state, making the reactant(s) less In a sense, if Near-Attack-Conformers are viewed as stable and thereby lowering the energy difference (indicated enzyme-stabilized ‘‘pre-transition-state’’ structures facing by red arrows) between strained reactant(s) and transition only a modest barrier to reaction, they might just as well be state. (Note: The terms ground-state destabilization (GSD) thought of as part of an ensemble of enzyme-stabilized and reactant-state destabilization (RSD) are interchange- transition states. Indeed, the smaller the barrier to reaction, able.) Some form of reactant distortion, bond strain, bond the more like a stabilized transition state would be an NAC. 30 Enzyme Kinetics
1.5.6 Acid/Base Catalysis intermediates confirms that significant advantages must be gained from their formation. Enzymes organize covalent As discussed in Chapter 2, formation of formal cationic or intermediate formation and turnover into discrete stages: anionic species, each respectively possessing fully devel- first, there is a nucleophilic stage, in which a catalytic oped electronic charges on the electron deficient or electron- functional group attacks the substrate to form a covalent rich atom, is a highly improbable event that necessarily bond; second, electrons are withdrawn by the now electro- results in a high DEact for reaction. Acids and bases often philic catalyst; and third, rupture of the covalent bond improve reactivity, and transfer of a proton (to the reactant by permits further reaction and regenerates the enzyme-based an active-site acid and from the reactant by an active-site nucleophile. The latter is typically the functional group of base) has the effect of lowering the energy of the transition a lysine, histidine, cysteine, aspartate, glutamate, and serine state, thereby reducing the activation energy DEact. An even residue within an enzyme’s active site. Many coenzymes greater enhancement is attained by the coordinated action of (e.g., pyridoxal 5-phosphate, biotin, lipoamide, thiamin an acid and base, as in the case of an active-site base attacking diphosphate, tetrahydrobiopterin, and even NADþ and FAD) a carbonyl, with attendant protonation by an active-site acid. also play essential roles in forming covalent intermediates. As discussed by Jencks (1963; 1969), formation of a covalent A intermediate per se is insufficient for highly effective covalent catalysis: beyond reacting rapidly, the active-site nucleophile H must yield a product that is itself highly reactive. He also O asserted that the chief advantage of enzymatic covalent catalysis R R S is that reaction mechanism can be organized in a manner that B: manages entropy changes (Jencks, 1975) while maintaining the group transfer potential of substrate-derived moieties (e.g., Scheme 1.9 phosphoryl groups, amino groups, nucleotidyl groups, etc.). That covalent catalysis requires a highly reactive active- The virtually simultaneous action of nucleophilic attack site nucleophile is well illustrated by the following two and protonation (Scheme 1.9) requires additional structural reactions. In the case of bacterial acetoacetate decarboxylase organization within the active site, requiring appropriate (Reaction: Acetoacetate # Acetone þ CO2), the group orientation of enzyme functional groups. If the energy headed by Frank Westheimer at Harvard University penalty for pre-organization is ‘‘paid’’ upon folding of the demonstrated that the enzyme exploits its surprisingly acidic nascent protein, much as suggested for electrostatic catal- e-amino group (pKa z 6.5), which is displaced by some four ysis, then acid/base catalysis requires no additional energy pH units from that of a typical lysine side-chain amino group. penalty for functional group orientation. Numerous observations confirm that complete proton O transfer need not occur. In fact, significant advantages O H O H2 2 accrue when a Brønsted acid partially donates a proton to H CCC C H3CCC C 3 (or when a Brønsted base partially abstracts a proton from) O O a reaction center (see Section 7.3.9: Brønsted Theory NH H2N Explains Important Aspects of Acid/Base Catalysis). Enz Finally, because the equilibrium between the active-site Enz CO base and its conjugate acid (or the active-site acid and its 2 O H conjugate base) is coupled to the catalytic cycle, enzyme H2 H CCC C H CC activity frequently displays a pH-dependence. Although 3 3 CH2 O steady-state kinetics is effective in discerning the pH NH N dependencies for Km, Vm,orVm/Km, the pH-dependence of Enz H elementary reactions rates is far more revealing. This is true Enz because steady-state parameters like Km, Vm,orVm/Km are Enz complex collections of elementary reaction rate constants, H3CCCH3 NH whereas fast kinetic studies directly establish the pH 2 OH H3CCCH3 dependence of individual elementary rate constants. N H
Enz O 1.5.7 Covalent Catalysis Scheme 1.10 Covalent catalysis refers to any catalytic rate enhancement gained from transient formation of covalent reaction inter- Shown in Scheme 1.10 is the likely mechanism showing mediates. That thousands of enzymes form covalent how the formation of imine and eneamine intermediates Chapter j 1 An Introduction to Enzyme Science 31 organizes stepwise decarboxylation (see Section 7.3.4 for synthesis of covalent intermediates, pyruvate dehydroge- a detailed discussion of this enzyme mechanism). Likewise, nase catalysis would presumably require additional steps as the research group headed by Daniel Santi at UC San well as the release of reactive intermediates. Francisco demonstrated that thymidylate synthase (Reac- Finally, in some enzyme-catalyzed reactions, forma- tion: dUMP þ Methylenetetrahydrofolate (CH2–H4Folate) tion of covalent intermediates also affords the opportu- # dTMP þ Dihydrofolate (H2Folate)) exploits covalent nity to control overall reaction stereochemistry. Two SN2 catalysis to activate dUMP for subsequent substitution reactions are needed to form and transfer a reactive (Carreras and Santi, 1995). After forming a reversible covalent intermediate, a scheme that results in overall ternary complex with its substrates, this synthase directs the retention of configuration. With direct in-line transfer, nucleophilic attack of its active-site thiol on C-6 of dUMP, however, only one SN2 reaction is needed, resulting in converting C-5 into a nucleophilic enol(ate) intermediate. overall inversion. Interestingly, for SN1 mechanisms, the Subsequent covalent bond formation ensues between that stereochemical outcome depends on how the carbenium site and the one-carbon unit (at C-11) of CH2–H4Folate, ion intermediate is intercepted. Because subsequent itself having been activated by formation of an N-5 iminium enzyme-catalyzed reactions within a metabolic pathway ion. Proton abstraction from the second key intermediate are often stereospecific, the stereochemical course of and b-elimination of H4Folate yields the exocyclic a preceding enzyme must be maintained, and a mecha- methylene intermediate. Hydride transfer from non- nism requiring covalent catalysis successfully fulfills this covalently bound H4Folate to the exocyclic methylene requirement. intermediate is followed by b-elimination of the enzyme, producing dTMP and H2Folate as well as regenerating the original active enzyme. 1.5.8 Transition-State Stabilization An added benefit of covalent catalysis is that reactive intermediates can be shuttled from one active site to another by Low-Barrier Hydrogen Bonds in multi-enzyme complexes. One example is trans- A special type of transition-state stabilization, first sug- carboxylase (Reaction: Methylmalonyl-CoA þ Pyruvate # gested by Schowen (1988) and promoted by Cleland and Propionyl-CoA þ Oxaloacetate), which catalyzes a multi- Kreevoy (1994), concerns the possibility that the protected site Ping Pong mechanism (Northrop, 1969). interior of certain active sites may favor formation of strong hydrogen bonds, known as low-barrier hydrogen bonds S (LBHBs). Unlike most other hydrogen bonds, which have H 2.9–3.3 A˚ distance between electronegative atoms, the O C 2 N N bond-length of low-barrier H-bonds is less than 2.5 A˚ . ENZYME NH Neutron diffraction experiments on crystalline compounds O O containing LBHBs indicate that the shared proton is diffusely distributed around the bond’s midpoint, a finding The transferred carboxyl group (shown in red) is carried suggesting that LBHBs exhibit covalent nature (see also from one active site (First Half-Reaction: Enz þ Methyl- Section 2.2.3). malonyl-CoA # Propionyl-CoA þ Enz–CO2 ) to a second Cleland, Frey and Gerlt (1998) suggested that low- active site (Second Half-Reaction: Enz–CO2 þ Pyruvate barrier hydrogen bonds may contribute upwards of five # Oxaloacetate þ Enz) by means of a long arm consisting orders of magnitude in rate acceleration in any enzymatic of a biotin cofactor (blue) covalently tethered to an reaction involving proton transfer from a general acid or to 3-amino group of transcarboxylase lysine residue (black). a base. Their argument goes as follows: LBHBs form when Another example is pyruvate dehydrogenase (PDH), the atoms sharing the proton have identical pKa values; so þ a multi-enzyme system that uses five cofactors: NAD , any equalization of their pKa values should enhance H-bond coenzyme A, thiamin diphosphate (TDP), lipoamide, and overlap, thus stabilizing the transition state and promoting FAD. PDH catalyzes the overall reaction of pyruvate with catalysis. Cleland and Kreevoy (1994) suggested that NADþ and coenzyme A to produce acetyl-CoA, NADH, LBHBs may provide up to 10–20 kcal/mol of transition- and CO2. PDH first catalyzes the TDP-dependent reaction state stabilization; however, model studies on LBHBs put of pyruvate with lipoamide to form S-acetyl-dihy- the value at 4–5 kcal/mol in dimethyl sulfoxide and 3–6 drolipoamide and CO2. Dihydrolipoamide S-acetyl- kcal/mol in tetrahydrofuran (Shan, Loh and Herschlag, transferase next catalyzes the reaction of S-acetyl- 1996). Usher et al. (1994) estimated the value to be nearer to dihydrolipoamide with coenzyme A to produce dihy- 2 kcal/mol, a value that comports with mutagenesis data drolipoamide and acetyl-CoA. Then the FAD-dependent (Fersht, 1987). Shurki et al. (2002) also questioned whether dihydrolipoamide dehydrogenase uses its active site to LBHBs can account for catalytic rate enhancements catalyze the reaction of dihydrolipoamide with NADþ to observed with protein enzymes. Paradoxically, Warshel produce lipoamide and NADH. Without the intervening et al. (2006) argue that, when consistently defined, 32 Enzyme Kinetics low-barrier hydrogen bonds are more apt to exert an anti- step-by-step through the catalytic cycle. As indicated in catalytic effect. Fig. 1.4, the same catalytic histidine residue acts as a: (a) general base by accepting the proton from the catalytic serine, thereby activating the latter’s nucleophilicity; (b) 1.5.9 Catalytic Facilitation by Metal Ions general acid by donating a proton to the nitrogen on the Although enzyme- and substrate-bound metal ions exert leaving group; and (c) general base that deprotonates and powerful electrostatic stabilization of transition states, activates the ‘‘hydrolytic’’ water. metal ions are known to facilitate catalysis in many other In the context of Hammes’ comments, it seems clear that ways. By taking advantage of the well-defined geometric conformational changes in chymotrypsin trigger changes in arrangement of their inner-coordination spheres, transition catalytic group reactivity and vice versa. Radisky et al. metal ions often serve as templates that hold and orient (2006), for example, found that atomic-resolution structures reactive molecules during one or more phases of the cata- of acyl-trypsin and enzyme-bound tetrahedral intermediate lytic reaction cycle. Metal ions are also highly versatile analogue, along with earlier structures for the Michaelis Lewis acids (i.e., electron-pair acceptors) that can alter the complex, provide evidence of subtle active-site adjustments reactivity of acidic and basic functional groups. Metal ions favoring the forward progress of the acylation reaction. It alter the pKas of bound substrates as well as bound water, should be emphasized that the energetics at each reaction thereby improving their tendency to react. Metal ions also stage can be finely tuned to facilitate catalysis without transiently switch oxidation states during catalysis, and in violating the constraint that an enzyme cannot alter the some cases, they achieve unusually reactive higher oxida- overall reaction’s equilibrium poise. tion states. Ca2þ and Mg2þ also bind to ATP4 to form Mutual cuing between catalyst and reactant also fits with CaATP2 and MgATP2 , thereby activating the latter action–reaction principles of classical mechanics. There- z toward nucleophilic agents (see Section 2.5: Metal Ions in fore, although E$X is almost universally employed to z Enzyme Active Sites). represent an enzyme-bound transition state, (E$X) is perhaps a more appropriate indicator that both the catalyst and substrate are mutually altered as they proceed through 1.5.10 Promotion of Catalysis via Enzyme each catalytic cycle. This effect represents an example of Conformational Flexibility thermodynamic reciprocity (i.e., a catalyst cannot affect the In seeking to summarize the mounting evidence for a role of reactant without the reactant affecting the catalyst). Use of $ z protein dynamics in enzyme catalysis, Hammes (2002) E X leaves a mistaken impression that only the substrate offered the following comments: reaches the activated complex or transition-state configu- ration. If this were the case, the intimacy of motions within When a substrate binds to an enzyme, it becomes an inte- an enzyme-substrate would be ignored. Writing the overall gral part of the macromolecule. The subsequent dynamics transition state as (E$X)z implies that enzyme and substrate of the macromolecular conformational changes are then jointly achieve transition-state intermediacy, a process the catalytic process itself. This view of catalysis means requiring simultaneous motions in reactant and enzyme. that the making and breaking of non-covalent bonds within Another way of explaining the comparatively large size the structure are part of the catalytic process, and that these of enzymes is that catalysis is a complex, multi-step process events can occur close to and far from the active site. The requiring an active-site environment that optimally stabi- advantage of having hundreds of intramolecular interac- tions dynamically involved in catalysis is that the ener- lizes multiple transition states, each associated with its own getics of the reaction can be easily manipulated to step. Conformational flexibility is apt to be a hallmark of produce catalysis, and extremely fine-tuning is provided effective multistage reaction catalysis and even suggests by hundreds of intramolecular interactions. This mecha- why protein enzymes are apt to be more highly perfected nism could be viewed as a ‘gear shift’ mechanism: the than nucleic acid enzymes. Benkovic, Hammes and conformational transitions are analogous to shifting gears, Hammes-Schiffer (2008) suggested that: and the interactions between the enzyme and substrate correspond to the gear coupling mechanism. Asking what Enzyme mechanisms should be viewed as catalytic ‘drives’ the reaction is not terribly meaningful, as the networks with multiple conformations that occur serially essence of cooperative processes is that many events are and in parallel in the mechanism. These coupled ensembles occurring essentially simultaneously. of conformations require a multi-dimensional standard free-energy surface that is very rugged, containing multiple Hammes’ cogent comments are tantamount to saying minima and transition states. that an enzyme creates a dynamic catalytic environment, one that promotes the trajectory of substrate to product by These features are shown in Fig. 1.7. As considered in way of one or more reaction intermediates. Much as well- Section 12.3, this concept was anticipated in the derivation of rehearsed actors cue each other, a succession of structural a single-molecule Michaelis-Menten equation by Kou et al. cues, each created as the enzyme and reactant proceed (2005) who present a virtually identical view of an enzyme Chapter j 1 An Introduction to Enzyme Science 33
1.5.11 Promotion of Catalysis via Force- Sensing and Force-Gated Mechanisms
G° As scientists, we should keep an open mind as to the possibility that we have been trapped into thinking that enzyme chemistry must operate by mechanisms that resemble those for gas-phase and solution-phase organo- chemical reactions. The chemistry within enzyme active sites may eventually prove to be fundamentally different. E+S All the enzymes that are known to catalyze some 10,000 to 20,000 different reactions in living organisms Reac constitute an infinitesimally small subset of 20500 possible tion Coordina polypeptides of molecular masses of 50 kDa or less. What te E+P may distinguish these 10,000 to 20,000 enzymes as the 500 Ensemble Conformations rarest of the rare among those 20 polypeptides is that each has its respective reaction trajectory already pro- FIGURE 1.7 Schematic representation of the standard free-energy grammed into its conformationally compliant structure. If landscape for a catalytic network of an enzyme reaction. The catalytic process is viewed as proceeding through a network consisting of a multi- this unique catalytic choreography avoids the nonproductive tude of conformations and numerous catalytic reaction cycles, each written molecular configurations inevitably made in gas-phase and horizontally as a reaction path (e.g.,E1 # A1 # B1 # # P1 # E1; solution-phase reactions, an enzyme may not require # # # # # # # # # # E2 A2 B2 P2 E2; and E3 A3 B3 P3 a significant energy input to populate productive configu- E3, etc.), where each species is connected vertically to its corresponding rations. For example, the observation that heating gas-phase conformer (e.g.,E1 # E2 # E3 # # En–1 # En). The result is a network similar to that shown in Scheme 12.5 describing the results of and solution-phase reactants increases molecular agitation single-molecular enzyme kinetic data. For simplicity, only one substrate and generally enhances reactivity is thought to be the S and one product P are shown. Note that enzyme conformational changes consequence of populating high-lying transition-state actually occur along both axes: (a) those changes along the reaction coordi- configurations needed to convert a reactant into its product. nate axis correspond to the environmental reorganization facilitating chem- Even so, heating of reactants also produces countless ical reaction; and (b) those changes occurring along the ‘‘ensemble conformations’’-axis represent the ensembles of configurations existing at nonproductive configurations, thereby greatly limiting the all stages along the reaction coordinate. Therefore, a plane parallel to the fraction of molecules that are appropriately oriented. Thus, axis labeled ensemble conformations bisects this catalytic ‘‘mountain while we now think in terms of reaction coordinate range’’ along the red mountaintop, with reactants E þ S are on one side diagrams resembling those for solution-phase models, of the plane and the products E þ P on the other. This free energy landscape enzymes may accelerate reactions in ways that are beyond thus illustrates the multiple populations of conformations, intermediates, and transition states. Strong coupling can occur between the reaction coor- our reckoning, simply because we may be incorrectly dinates and the conformation ensembles (i.e., the reaction paths can slide perpetuating the notion that enzyme mechanisms are ‘‘not along and between both coordinates). For real enzymes, the number of different, just better.’’ In carrying out covalent bond trans- maxima and minima along the coordinates is expected to be greater than formations, most enzymes may be acting in a manner that is shown. The dominant catalytic pathways will be altered by external condi- functionally complementary to the action of mecha- tions and protein mutations. Figure (originally created by S. J. Edwards) and legend adapted from Benkovic, Hammes and Hammes Schiffer noenzymes, meaning that they are programmed to make (2008) with permission of the authors and the publisher. directed motions via precise noncovalent bond rearrange- ments of the active site, and perhaps even the entire protein, so as to convert the covalent bonds of the substrate into the operating by a catalytic network with multiple conformations covalent bonds of the product. Simply put, although (see also Section 3.8: Transition State Theory). enzymes undergo the same types of reactions and also Finally, although some nucleic acids serve as biological likewise form many of the same types of intermediates as catalysts, they are feeble in comparison to protein enzymes those observed in cognate gas-phase and solution-phase (Purich, 2005). One may therefore speculate that the far organochemical, enzymes may not be confined by the rules greater conformational flexibility of proteins and their of physical organic chemistry, at least those rules gleaned consequentially higher catalytic efficiency may have been from studies of corresponding gas-phase and solution-phase major driving forces in the early evolution away from reactions. nucleic acid-based catalysts in favor of the far more versa- There is reasonably general recognition that no single tile protein-based catalysts. Organisms managing to cata- property of an enzyme likely to underlie the origin of enzyme lyze a reaction much faster than a rival should have enjoyed rate enhancements and that each enzyme may exploit a substantial advantage. Retention of some roles for cata- more than one in the course of its catalysis. What becomes lytic RNA also suggests that rate enhancement may not be evident is that many of the above ideas converge, if one as important for the catalysis of certain reactions. conceives of all enzymes – not just mechanoenzymes – as 34 Enzyme Kinetics force-generating and force-sensing molecular machines that sections describe areas where sustained inquiry is apt to are exquisitely well designed to: (a) recognize and bind reap great rewards. specific substrates; (b) avoid unduly tight binding inter- actions with the substrate as well as catalytic cycle inter- mediates and reaction products; (c) bring about 1.6.1 We Need Better Methods for Analyzing conformational changes that continually re-position active- Enzyme Dynamics to Understand the site groups as the catalytic cycle proceeds; and (d) promote Detailed Mutual Changes in Both Substrate enzyme evolution and perfection by adjusting the energetic landscape to create alternative mechanochemical pathways and Enzyme During Catalysis for catalysis. In this way, some steps within a catalytic As noted earlier in this chapter, the distinction between reaction cycle may be viewed as force-gated conformational chemical kinetics and chemical dynamics is that the former changes that constantly readjust catalytic determinants focuses on the measurement of reactivity (i.e., reaction within the active-site to optimize the local push-pull force rates) with an emphasis on bond-making/breaking mecha- balance between the enzyme and various forms of bound nisms of chemical transformations, whereas the latter refers reactant. All reaction coordinate diagrams plot DG (or to the atomic and molecular motions that influence reac- change in potential energy DU) on the ordinate versus tivity and stability. While chemical intuition guides the ‘‘Reaction Progress’’ (often indicated as some inter-atomic notion that internal enzyme flexibility is essential for distance, say d, representing a bond making or bond- activity, the nature of catalytic motions is poorly under- breaking event) on the abscissa. The slope DG/Dd (or DU/ stood. A longstanding question about biological catalysis Dd) corresponds to a pushing or pulling force F that is concerns the functional coupling of reactant motions to the mutually experienced by enzyme and its bound reactant(s) as enzyme’s local conformational dynamics in various Enzy- they jointly approach and surmount the transition state. me$Substrate, Enzyme$Intermediate, and Enzyme$Product Such ideas also fit with the universal occurrence of complexes. The great speed of catalysis has been a major domains and motifs that are connected by hinges and joints, obstacle for ‘‘on-the-fly’’ analysis of conformational where forces can be localized and/or managed (Williams, dynamics. Because the time-scale of each catalytic reaction 1993). In this respect, an enzyme’s mechanochemical cycle sets the longest lifetime of any intermediate, an properties appear to be a natural complement to its bond- enzymic reaction proceeding at a rate of 5,000 cycles/s has breaking/making properties, as illustrated by the capacity of a 0.2 millisec catalytic cycle-time, one that is too short for ATP synthase to use ATP hydrolysis to drive the confor- most techniques that can detect individual residue side- mational changes that energize transmembrane proton chain motions. gradients or that use the latter to drive ATP synthesis There is in fact mounting evidence that protein dynamics (Purich, 2001). may play a central role in enzymatic catalysis, well beyond the standard models of loop motions that help to hold substrate(s) within a desolvated active site (Hentzler-Wild- 1.6 PROSPECTS FOR ENZYME SCIENCE man and Kern, 2007). Directed motions of the enzyme per se may be coupled to the catalytic mechanism, especially in Predicting the likely direction that a scientific field will those cases where hydrogen tunneling seems to be operating take is an inherently hazardous enterprise, mainly because (Basran, Sutcliff and Scrutton, 1999). The basic idea is that field-changing intellectual realization occurs in bursts and rate-promoting vibrations are intrinsic motions of the often exploits completely unanticipated opportunities. A protein catalyst that form a dynamic matrix surrounding seemingly insignificant advance in one scientific discipline the substrate, and that these vibrational modes can alter the may also trigger a breakthrough in another field. What is geometry of the bonding barrier(s) to chemical reaction. self-evident is that enzymes have been bestowed with When viewed from this perspective, the defining nature of a special status in the chemical sciences, and for nearly a promoting vibration is to be found in the nature of the two centuries, the chemical, biochemical, and physiologic coupling of that protein matrix motion to the reaction actions of enzymes have continually piqued the intellec- coordinate (Caratzoulas, Mincer and Schwartz, 2002). tual curiosity of highly creative individuals. By enriching Antoniou et al. (2002) described how a catalysis-promoting our understanding of enzymes and the physiologic vibration within the enzyme may be coupled to a vibrational behavior, many enzymologists have even earned Nobel mode of a reactant proceeding along the reaction coordi- Prizes (Table 1.4). nate. Their view is that evolution created a protein structure One may therefore assert that enzyme science will that moves in such a way that lowers and narrows the barrier surely enjoy a brilliant future, and it’s safe to assume that to reaction. This lowering of the barrier is not merely this intellectually stimulating, and yet immensely prac- a statistical lowering of a potential of mean force through tical, enterprise will doubtlessly prosper from the dev- the release of binding energy; rather, the enzyme is believed elopment of new kinetic approaches. The following to use highly directed energy in the form of a vibration Chapter j 1 An Introduction to Enzyme Science 35 acting in a specific direction. It is believed that rate- probed motions on time-scales shorter than ~100 ps, and the promoting vibrations within protein catalysts have IN16 spectrometer extended the time-scale to ~5 ns. Their 150-cm 1 frequencies, corresponding to vibrations on the results demonstrated a marked dependence on the time- sub-picosecond time-scale. Because enzyme catalysis scale of the temperature profile of the mean square occurs with frequencies of 104–107 s 1, there is an unex- displacement. Several dynamical transitions were observed plained disparity in vibrational and catalytic time-scales. in the slower dynamics. Comparison with the temperature Many hundreds of thousands of these rate promoting profile of the activity of the enzyme in the same solvent vibrations occur over the time needed for a single catalytic reveals dynamical transitions having no effect on GDH round. Because vibrational energy obeys the Boltzmann function. Representing the first assessment of the global distribution (see Section 3.6: Thermal Energy: The Boltz- dynamics of an active enzyme measured under similar mann Distribution Law), it’s possible that a rare (and hence conditions over a range of time-scales, these studies suggest substantially more energetic) vibration may be needed to that anharmonic, picosecond motions are not required at all trigger catalysis. Finally, Caserta and Cervigni (1974) temperatures for the enzyme rate-limiting step. The authors offered a more rudimentary suggestion that nonetheless suggest that anharmonic fast motions are not necessarily postulated electron induced, selective amplification of low- coupled to the much slower motions describing transitions frequency vibrational waves in the enzyme, such that these along the enzyme reaction coordinate. They caution, vibrations are coupled to a susceptible region of the however, that the neutron technique reveals average substrate, with consequential lowering of the activation dynamics, and it is conceivable that functionally important energy. fast motions may occur locally in the protein at the active Although hydrogen-deuterium and disulfide-trapping site, but below noise levels. techniques can clearly detect 15-A˚ protein motions on Eisenmesser et al. (2002) used magnetic resonance the millisecond time-scale (Careaga and Falke, 1992; spectroscopy to analyze conformational exchange in the Englander and Kallenbach, 1983; Falke and Koshland, reaction catalyzed by prolyl-peptidyl isomerase (Reaction: 1987; Huyghues-Despointes et al., 2001), these methods cis-X–Pro Isomer # trans-X–Pro Isomer), also known as are uninformative about faster processes. For example, the cyclophyllin A: hydrogen deuterium exchange technique, which quantifies the time-course for the release of protons bound up in O H O a-helix and b-sheet structures, is incapable of providing N H H N R 2 such information on a sub-millisecond time-scale. Of N C C N R H R2 particular interest is whether picosecond and nanosecond 1 H R H O 1 O time-scale structural fluctuations are coupled to the structural changes associated with the catalytic rate- peptidyl-trans-proline peptidyl-trans-proline limiting step, the latter typically occurring on the micro- The simplest catalytic cycle consistent with known second-to-millisecond time-scale (Daniel et al., 1999). An catalytic properties is shown in Scheme 1.11, with three important question is whether the fast motions need to be microscopic reaction steps: anharmonic, such that picosecond-to-nanosecond motions in the protein may be needed to permit slower micro- second millisecond dynamics across the highest-energy E reaction barrier. ktrans,on kcis,on The first parallel comparison of the activity and k k dynamics of glutamate dehydrogenase (GDH), as probed trans,off cis,off picosecond time-scale motions, showed no deviation from kc-to-t,cat Arrhenius behavior through the dynamical transition E-Prolinetrans E-Prolinecis k (Daniel et al., 1998). The experiments were performed in t-to-c,cat a 70% vol/vol methanol/water cryosolvent in which the Scheme 1.11 enzyme is active and stable. For the thermophilic microbial GDH operating near 350 K, the turnover number of the where Ktrans,D ¼ ktrans,off/ktrans,on and Kcis,D ¼ kcis,off/kcis,on. enzyme is ~1500 s 1 at ~350 K, and in fully deuterated Eisenmesser et al. (2002) conducted 15N spin relaxation cryosolvent at 220 K, the turnover number is ~0.01 s 1. experiments in the absence and presence of the substrate Their results indicated that over the 190–220 K temperature N-Succinyl-L-Ala-L-Phe-L-Pro-4-NA. Chemical-shift mapping range, the enzyme’s rate-limiting step(s) is(are) unaffected with 15N yields a single resonance (or a single peak in a two- by picosecond protein motions. To extend the time-scale dimensional NMR spectrum) for each amide bond. By problem, Daniel et al. (1999) used advanced neutron scat- changing the relaxation delay time, they determined the tering spectrometers to compare the temperature depen- transverse relaxation rate constant R2, which obeys the dence of GDH activity and dynamics. The IN6 spectrometer relation: R2 ¼ R20 þ Rex. The latter represents the exchange 36 Enzyme Kinetics
contribution to R2 and provides information about the relevant motions that occur on the microsecond-to-milli- second time-scale. To separate the effects of binding from cis-trans isomerization, the authors characterized substrate concentration-dependent changes in R2. The relative contributions to R2 from exchange due to binding and cis- trans isomerization exhibited different dependencies on 2 substrate concentration. For most residues, Rex z pApBdv / kex where pA and pB are the fractional populations of free enzyme and the bound states, dv is the chemical shift difference between E and SE$Si, and kex is the exchange rate. As substrate concentration is increased, Rex therefore rises and then falls; maximal chemical exchange occurs at intermediate substrate concentrations, where Efree z (E$Scis þ E$Strans). The observed rate behavior fits with the presence of significant concentrations of the three protein 15 forms (i.e.,E,E$Scis, and E$Strans). When plotted as the N R2 relaxation rate constant versus residue number (Fig. 1.8), it became clear that certain regions in the enzyme exhibited changes in R2 due to steady-state catalytic turnover. By nonlinear regression analysis of the exchange contribution to R2 for those residues sensing substrate binding and catalysis, the authors obtained Km values ranging from 0.95 FIGURE 1.8 Residues in cyclophyllin A exhibiting microsecond time- 1 to 1.2 mM, and koff values of 10,700 to 14,800 s . scale dynamics during catalysis. Structures of the enzyme-bound cis and Based on other quantitative estimates of the rate trans conformations of the substrate N-Succinyl-Ala-Phe-Pro-4-NA constants for the protein’s structural dynamics, the (green) bound to the enzyme (including expanded views shown at right), based on the X-ray structure of CypA complexed to the cis form of authors reached the important conclusion that areas N-Succinyl-Ala-Phe-Pro-4-NA (1RMH) (Zhau and Ke, 1996). CypA resi- 1 3 9 around residues 55, 82, 10 –10 , and 10 playarolein dues with chemical exchange in both the presence and absence of substrate substrate binding at or near the diffusion limit. After the are color-coded in blue (namely Phe-67, Gln-71, Gly-74, Ser-77, and Ser- substrate is bound, the enzyme catalyzes a 180 -rotation 100). Residues in red exhibit chemical exchange only during turnover of the prolyl peptide bond, and the substrate tail on the (Arg-55, Lys-82, Leu-98, Ser-99, Ala-101, Gln-102, Ala-103, and Gly- 109). Residues shown in magenta (Thr-68 and Gly-72) exhibit chemical C-terminal side with respect to the prolyl residue is exchange in the absence of the substrate but increase in its presence. viewed as swinging around to make contact with the CypA catalyzes prolyl isomerization by rotating the C-terminal part of enzyme near residues 98 and 99. Meanwhile, the sub- the prolyl peptide bond by 180 to produce the trans conformation of strate’s N-terminal tail stays fixed, allowing the E$S the substrate. In this model, the observed exchange dynamics of residues complex to remain intact, despite substantial rearrange- in strand-5 of the enzyme can be explained. Reproduced from Eisenmesser et al. (2002) with the permission of the authors and the American Associ- ments during the cis-trans isomerization at a rate of ation for the Advancement of Science. 9,000 s 1. Most notably, motions of the substrate and the enzyme coincide, and the catalytic Arg-55 also moves withthesamerateconstant. Earlier two-dimensional heteronuclear (1H–15N) nuclear The beauty of this investigation on cyclophyllin A magnetic relaxation studies suggested that the dihydrofolate catalysis is that Eisenmesser et al. (2002) succeeded in reductase$dihydrofolate complex exhibits a diverse range of identifying those regions of the enzyme whose dynamics backbone fluctuations on the psec-to-nsec time-scale match the essential enzyme kinetics of catalysis. They also (Epstein, Benkovic and Wright, 1995). To assess whether mapped the microsecond time-scale dynamics to specific these dynamical features influence Michaelis complex regions of the cis-trans isomerase. Despite the fact that formation, Miller and Benkovic (1998) used mutagenesis additional analysis is needed to define the motions during and kinetic measurements to assess the role of the strictly the actual catalytic event, their systematic approach defined conserved residue Gly-121, which displays large-amplitude dynamic ‘‘hot spots’’ during catalysis and revealed that the backbone motions on the nanosecond time-scale. Deletion time-scales for protein dynamics coincide with those for of Gly-121 dramatically reduces the hydride transfer rate by substrate turnover. Finally, Bosco, Eisenmesser and Kern 550 times; there is also a 20-times decrease in NADPH (2002) also described CypA’s catalytic action on Pro-90 in cofactor binding affinity and a 7-fold decrease for NADPþ the HIV capsid protein. Their work is the first documented relative to wild type. Insertion mutations significantly case of catalyzed cis-trans isomerization on a prolyl residue decreased both substrate and cofactor binding. Their results within a natively folded protein substrate. suggest that distant residues, such as Gly-121 in DHFR, Chapter j 1 An Introduction to Enzyme Science 37 may influence the formation of liganded complexes as well at around 20,000 to 30,000 for all species, not counting many as the proper orientation of substrate and cofactor during the billions of largely inconsequential, but naturally occurring catalytic cycle. amino acid substitutions; however great the number of such Finally, it is also worthwhile to ponder a related question: naturally occurring enzymes, the combinatorial variability of Why are enzymes so large? Aside from the structural polypeptides, with say 400 residues, would be an astonish- complexity of allosteric enzymes, the most common answer ingly great (~20400). Present day ideas about critical enzyme is that most enzymes are made up of domains and motifs, residues and the focused flow of energy within proteins are the binding properties of which have been honed through best characterized for redox proteins like the cytochromes, Natural Selection. In numerous lectures on chemical and rhodopsin, green fluorescent protein, as well as photosyn- enzyme catalysis, the late Daniel Koshland was fond of thetic reaction centers. Even then, the actual energy-flow comparing a hydroxide ion to a hand-drill and an enzyme to pathways are at best sketchy. a milling machine. His point was that enzyme catalysis As reviewed by Leitner (2008), energy flow within almost certainly requires highly precise interactions of an a protein may be treated as a percolation process involving enzyme with its substrate(s). In the context of transition- a network of sites, some resulting in fast transport when state stabilization, it is reasonable to anticipate that a strong distant points are directly connected by energy-flow chan- and exact fit of enzyme and substrate within the (E$X)z nels, with others exhibiting slow transport along numerous transition-state complex is critically important. Similarly, pathways that most often reach dead ends. This connection binding energy is an essential ingredient for ground-state can be made more precise by comparing statistically energy destabilization. From the perspective of enzymes as force- flow in proteins with flow related to the nature and density actuated catalytic devices, however, the oil-like properties of a protein’s vibrational states. Energy transfer can occur as of the hydrophobic cores of globular enzymes may absorb, molecular vibrations or by dipole–dipole interactions in redirect, and align forces imparted by thermal energy with photoexcited states. The former, which is limited by the respect to the trajectory of E$S along the reaction coordi- speed of sound and is most frequently carried by the rela- nate. In this respect, domains may also focus these forces at tively low-frequency modes of a protein, occurs on the order critical stages within the catalytic cycle, much like lattice of 10 A˚ /psec 1. With a mean free path on the order of 1 A˚ , dislocations are thought to facilitate heterogeneous catalysis the shortest time over which diffusion can be observed is on metal surfaces. While the breaking of discrete chemical around 0.1 picoseconds. For proteins consisting of 100 bonds occurs on the picosecond time-scale, protein residues, energy diffuses from the interior to the surface in conformation changes occur on the same nanosecond-to- a few picoseconds, so vibrational energy flow in proteins microsecond time-scale, as is observed for enzyme catal- exhibits anomalous subdiffusion, with times of approxi- ysis. Any force F exerted over a distance Dx along the mately 0.1 picoseconds. Although the nature of fluorescence reaction coordinate should have the effect of reducing the resonance energy transfer (FRET) will be described in zero-force DEact,0 value to the effective activation energy Section 4.5.6, it is sufficient here to say that the efficiency of DEact,effective, such that DEact,effective ¼ DEact,0 – FDx.A FRET depends on the relative orientation of the donor and large protein may even have the effect of increasing the acceptor moieties as well as the distance between them, magnitude of Dx, thereby further reducing the effective with the latter imposing an inverse sixth power dependence activation energy. on that distance. When the acceptor is photo-emissive, a photon will be emitted after a red-shift (i.e., the donor will absorb shorter wavelength light than that emitted by the 1.6.2 We Need New Approaches for acceptor), and the timescale will depend mainly on the Determining the Channels Allowing Energy fluorescence lifetime of the acceptor. When the acceptor is a quencher, one must entertain the possibility that the Flow During Enzyme Catalysis resulting thermal energy of the excited-state acceptor may A related outstanding problem in enzyme science concerns conceivably be channeled into discrete vibrational modes the if’s, where’s, when’s, and how’s of energy flow within and/or conformational changes. enzyme molecules during catalysis. For nearly a century, the Little solid information exists concerning the internal main approach of enzyme chemists has been the determi- transmission of energy within enzymes, and even less is nation of the enzyme-catalyzed transformations of substrates known for enzymes during catalysis. The extreme celerity to intermediates and thence to products, without due of enzymic catalysis imposes both technical limitations on consideration of how enzymes might manipulate the flow of the detection and quantification of transient changes in energy to achieve their enormous catalytic rate enhance- enzyme structure as well as substantial uncertainties ments. The origin of that energy and its detailed path(s) regarding the positions, motions, and momenta of critical within an enzyme molecule could, in principle, explain why catalytic residues. Even a fraction of an A˚ ngstro¨minthe Nature relies on such a small number of proteins for catal- position of a catalytic functional group could easily spell the ysis. Current estimates put the number of different enzymes difference between a poor and highly efficient catalyst. 38 Enzyme Kinetics
1.6.3 We Need Additional Probes independent MD simulations provide a very useful cross- of Enzyme Catalysis validation method for highly mobile regions that exhibit poorly defined experimental density. Likewise, informa- To define the mechanisms of energase-type mechano- tion from Laue difference maps provides information chemical reactions, one must learn how the DGhydrolysis (or about substrate conformation and interactions that greatly DGelectron-transport) drives protein transitions between non- facilitate MD simulations. covalent substrate- and product-like interaction states. In a truly formidable undertaking, Schmidt et al. Although modern protein science seeks to understand how (2003) successfully determined the number of authentic conformational energy is generated, stored, and managed, late-stage photo-cycle intermediates of PYP, the 14-kDa there are as yet no rules for predicting likely reaction photoactive yellow protein from the purple eubacterium intermediates and transition states in energase catalysis. Ectothiorhodospira halophila. PYP possesses a 4- From this perspective, the task of elucidating energase hydroxycinnamic acid chromophore linked as a thiolester mechanisms represents a monumental challenge for enzy- to Cys-69. Its 446-nm lmax matches the action spectrum mologists and structural biologists alike. Although fluores- for negative phototaxis, suggesting that PYP is the cence and Fo¨rster resonance energy transfer are powerful primary cytoplasmic blue-light photoreceptor for this tools for detecting and quantifying noncovalent interactions process. Schmidt et al. (2003) used laser light absorption and conformational transitions, A˚ ngstro¨m-scale resolution to trigger the series of room temperature chemical reac- is needed to unambiguously define structural alterations that tions in PYP crystals, and they then employed the Laue attend enzyme catalysis. Enzyme science will therefore diffraction technique (see 10.6.1: Flash Photolysis)to benefit enormously by the development of additional determine atomic structures of PYP after a laser-to-X-ray spectroscopic and crystallographic tools capable of interval of 5 ms, 9 ms, 20 ms, 51 ms, 125 ms, 250 ms, 500 discerning the small structural changes occurring in the ms, 850 ms,1ms,2ms,7ms,15ms,30ms,or100ms. enzyme during catalysis. When coupled with appropriate They applied singular value decomposition (SVD) to the computer-based modeling of enzyme interactions with series of experimental, time-dependent difference maps. substrates and inhibitors, a fuller picture of catalysis should This approach allowed them to evaluate rival chemical emerge. kinetic mechanisms and to arrive at a self-consistent The promise of time-resolved X-ray crystallography mechanism through their analysis of a set of time- must not be underestimated. Of particular note is the Laue dependent difference electron density maps spanning the diffraction method, which uses polychromatic X-rays time range from 5 ms to 100 ms. Successful fit of (typically l < 2.0 A˚ ) to collect sufficient structural data to exponentials to right singular vectors derived from compute a series of images on a short time-scale (Moffat, a singular value decomposition of the difference maps 2001). Although Laue diffraction and computational demonstrates that a chemical kinetic mechanism holds, molecular dynamics (MD) were developed as independent and that structurally distinct intermediates exist. ways to visualize and assess transient structural states, Schmidt et al. (2003) identified two time-independent their combined use may allow mutual refinement of difference maps, from which they refined the structures of computational MD simulations of Michaelis complexes the corresponding intermediates, thereby demonstrating and difference Fourier electron density maps obtained in how structures associated with intermediate states can be Laue experiments. Because a realistic molecular dynamics extracted from the experimental, time-dependent crystallo- study of a 50-kDa protein requires one to determine the graphic data. Stoichiometric and structural constraints 15 positions of ~10,000 atoms, every 10 seconds, large- allowed them to exclude one kinetic mechanism proposed scale MD simulations necessarily create huge data sets. for the photocycle but retain other plausible candidate The technique known as Principal Component Analysis is kinetic mechanisms. Thus, despite the fact that some might a mathematical tool for detecting correlations in large data justifiably quarrel with this author’s views as to whether sets. By expressing a molecular dynamics trajectory as a 446-nm photon is truly a substrate or whether the PYP a linear combination of principal components, the back- photocycle is catalytic, the approach taken by Schmidt et al. ground atomic fluctuations (i.e., thermal noise) are elimi- (2003) represents a bench-mark in pioneering efforts to nated, affording a better view of the protein’s collective analyze the time-evolution of an enzyme’s structure during motions (Balsera et al., 1996; Hayward, Kitaom and Go, catalysis. 1994; Mongan, 2004). When combined with appropriate physical models for protein motion, PCA can help one to detect genuine conformational changes. For example, 1.6.4 We Need to Learn How Proteins Fold mutual use of MD and crystallographic refinement allowed and How to Manipulate Protein Stability Stoddard, Dean and Bash (1996) to assign a number of Determining how proteins fold is also an enterprise of additional contacts and features for hydride transfer by central significance to enzymology, both with respect to isocitrate dehydrogenase. They reported that unrestrained how unfolded polypeptide chains self-organize to form Chapter j 1 An Introduction to Enzyme Science 39 active catalysts and how molecular chaperonins facilitate Directed evolution of novel,9 catalytically proficient such processes. The challenge is to conceive of and execute enzymes is quickly emerging as a powerful new theme in experiments that reveal the time-evolution of evanescent enzyme science. Biochemists are seeking to modify short-, medium- and long-range structures adopted by substrate recognition, to eliminate side-reactions, to form a protein during its folding and to develop adequate theories specific products, and to increase catalytic turnover rates. and simulation algorithms that capture essential features of Such efforts have traditionally been limited by the selection the folding process. Given the fact that folding can now be (or screening) method. In vivo selections are usually viewed as the consequence of a massive, parallel ‘‘diffu- restricted to identifying properties affecting the viability of sional’’ search of n-dimensional conformational space, the the organism, and full exploitation of these approaches is idea that discrete intermediates accumulate would imply often compromised by the complex nature of a living cell’s that there are kinetically significant bottlenecks in the intracellular environment and the need to transform that folding process. In their remarkable paper, Laurents and cell’s gene-library. Typically, 103–105 clone libraries are Baldwin (1998) discuss how the image of the transition state screened in a plate assay using a fluorogenic or chromo- has changed from a unique species (with a strained genic substrate to identify a few colonies of interest. To alter configuration and a correspondingly high free energy) to enzyme enantiomeric specificity for eventual use in asym- a more ordinary folding intermediate reflecting a balance metric organic synthesis, Reetz et al. (1997) proposed between limited conformational entropy and stabilizing a general approach that does not require any knowledge of contact places. As they explain, evidence for a broad tran- the structure or the mechanism of the enzyme, namely in sition barrier comes from studies showing that mutations vitro evolution using a combination of random gene muta- can change the position of the barrier. Controversy remains genesis by error-prone PCR (Leung, Chen and Goeddel, as to whether populated folding intermediates (i.e., those at 1989) and subsequent expression and high-throughput detectable concentrations) are productive ‘‘on-pathway’’ screening. To achieve error-prone Polymerase Chain intermediates or ‘‘dead-end’’ traps. Another confounding Reaction (or epPCR), the reaction conditions are varied issue concerns the generalizability of folding rules discov- empirically to reduce Taq polymerase fidelity during DNA ered to govern a particular protein. While these topics lie amplification, thereby causing base substitutions resulting well beyond the scope of this monograph, readers should in one, two, three, or even more amino acid substitutions in consult Dobson and Fersht (1995), Fersht (1998), and the encoded protein. Reetz (2004) discussed the scope and Richards et al. (2000). limitations of directed mutagenesis approaches, including the prospect of obtaining stereoselective hybrid catalysts composed of robust protein hosts in which transition metal 1.6.5 We Need to Develop a Deeper centers have been implanted. Some efforts have focused on using in vitro compartmentalization (IVC), an ingenious Understanding of Substrate Specificity approach wherein a reaction assay solution can be Understanding enzyme specificity remains an enormous unfulfilled challenge for structural biologists and enzyme chemists alike. Learning the rules governing substrate 9 specificity is essential in efforts to craft new metabolic When biochemists most often use the word ‘‘novel’’ to describe a substance, reaction, enzyme, etc., they are indicating that, to the pathways – a task of ever-greater significance in the design best of their knowledge, no such biochemical substance or reaction of microorganisms tailored to produce new plastics, has been previously reported. From a biological perspective, such renewable fuels, and novel therapeutics. Enzymes of 50- substances, reactions, enzymes, etc., are not new inasmuch as they kDa molecular mass have a molecular volume of ~100 nm3, have presumably been essential components for a long time. Given and their active sites are located in ~1-nm3 clefts and the introduction of manmade chemical substances into the environment for nearly two centuries, however, there is an increased crevices. In a sense, the complex, self-adaptive chemical likelihood for inadvertent evolution to give rise to a truly novel process that we call Life is only possible because each of enzymatic activity. A case in point is bacterial phosphotriesterase, 3 these 1-nm clefts and crevices exhibits a limited repertoire a microbial enzyme that catalyzes the hydrolysis of a broad range of of bio-specific interactions. Most active sites bind substrates phosphotriester substrates, including the neurotoxic cholinesterase and/or coenzyme with a combined molecular weight of inhibitors paraoxon (diethyl p-nitrophenyl-phosphate) and parathion (diethyl p-nitrophenyl-thiophosphate). As discussed by Shim, Hong 800–1,200 Daltons. The challenge of understanding enzyme and Rauschel (1998), the rarity of naturally occurring phosphotriester specificity not only speaks to the need for high-resolution substrates suggests that phosphotriesterase catalysis may be truly enzyme structures but also for kinetic data indicating how novel and that no such activity occurred prior to the introduction of subtle changes in enzyme structure determine interactions these agents into the environment. Biochemists are also interested in with substrates and inhibitors. If generalizable rules for directed enzyme evolution as a way to create new metabolic pathways or to improve chemical syntheses. Efforts to modify the enzyme specificity can be discovered, it should be possible chemical and/or kinetic properties of enzymes or to make catalysts to rebuild and/or remodel active sites to accommodate new from previously non-catalytic proteins and nucleic acids also raise the substances as substrates. likelihood for observing truly novel enzymatic activities. 40 Enzyme Kinetics partitioned into microscopic compartments, each of only ~5 An intriguing case of substrate specificity is the fL, by forming water-in-oil emulsions. In this way, a 50-mL D-ribulose-1,5-bisphosphate carboxylase/oxygenase, the 10 reaction volume can be dispersed into 10 physically iso- CO2-fixing enzyme that exhibits relatively slow catalysis lated, aqueous compartments, allowing for the selection of attributed to the need to discriminate between its substrates many genes and making the system highly sensitive and CO2 and O2. Tcherkez, Farquhar and Andrews (2006) economical. Tawfik and Griffiths (1998) and Lee, Tawfik argued that these characteristics arise from difficulty in and Griffiths (2002) demonstrated the feasibility of using specific binding of the structurally featureless CO2 mole- IVC to select DNA methyltransferases. Likewise, Levy, cule, forcing substrate specificity for CO2 versus O2 to be Griswold and Ellington (2005) used a compartmentalized in determined later (i.e., in the transition state). They suggest vitro selection method to directly select for ligase ribozymes that natural selection for greater CO2/O2 discrimination, in that are capable of acting on and turning over separable response to reducing atmospheric [CO2]/[O2] concentration oligonucleotide substrates. Starting from a degenerate pool, ratios, resulted in a transition state for CO2 addition that they selected a trans-acting variant of the Bartel class I resembles a carboxylate group. This adaptation maximizes ligase that statistically was likely to be the only active structural differences between transition states for variant in the starting pool, and isolation of this sequence carboxylation and oxygenation. However, the resulting from the population suggests that this selection method is increased similarity between the structure of the carboxyl- extremely robust at selecting optimal ribozymes. ation transition state and its carboxyketone product As a concrete example of a directed evolution experiment, exposes the carboxyketone to the strong binding needed to consider the work of Griffiths and Tawflik (2003) on the stabilize the transition state, causing the carboxyketone to selection of a high-kcat phosphotriesterase with turnover rates bind so tightly that its cleavage to products is slowed. >105 s 1, some 63 higher the wild-type enzyme. Mutant Tcherkez, Farquhar and Andrews (2006) suggested that enzymes were selected from a library of 3.4 107 mutated such apparent compromises in catalytic efficiency for the phosphotriesterase genes using the ingenious strategy of sake of specificity represent a new type of evolutionarily linking genotype and phenotype by means of in vitro perfected enzyme. compartmentalization (IVC) in water-in-oil emulsions. First, Substrate specificity also reinforces the idea that microbeads, each displaying a single gene and multiple copies enzymes are ideally suited for the synthesis and/or derivi- of the encoded protein, were formed by compartmentalized in tization of drugs. Consider, for example, the studies of vitro translation. To select for catalytic properties, the Khmelnitsky et al. (1997) focusing on the synthesis of microbeads were re-emulsified in a reaction buffer containing water-soluble forms of paclitaxel (taxol), the potent anti- a soluble substrate, and the product and any unreacted cancer drug that binds selectively to assembled micro- substrate were coupled to the beads when the reaction rate tubules. Scheme 1.12 shows that in the absence of any assay was complete. Product-coated beads, displaying active selective functional group protection, these investigators enzymes and the genes that encode them, were detected with identified a two-step enzymatic process for selective acyl- anti-product antibodies and selected using flow cytometry. ation and deacylation. With this completely in vitro approach, Griffiths and Tawflik There are two potentially reactive hydroxyl groups (2003) were able to select for substrate recognition, product (marked in red), but thermolysin selectively transfers the formation, rate acceleration and turnover. adipoyl moiety to only one, thereby preventing loss of Kim et al. (2001) simultaneously incorporated and biological activity by modification of the taxane ring. adjusted functional elements within an existing enzyme by Likewise, only one of the two ester-linkages (marked in inserting, deleting, and substituting several active-site loops, blue) is cleaved by the fungal lipase. Notice that both followed by fine-tuning of catalytic properties by means of reactions occur in polar organic solvents. site-directed point mutation. They successfully introduced There is also good reason to believe that biochemists b-lactamase activity into the ab/ba-metallohydrolase scaf- have not as yet identified all of the physiologically fold of glyoxalase II, and the re-engineered enzyme lost its significant ligands – even for those enzymes already original activity and gained the ability to catalyze the thought to be well characterized. The search for enzyme hydrolysis of cefotaxime with a (kcat/Km)app value of 1.8 regulatory molecules is often hit-or-miss, as evidenced by 102 M 1 s 1. While this specificity constant value is rather the serendipitous discovery of the pivotally important low, Escherichia coli containing the redesigned enzyme allosteric effector Fructose-2, 6-P2 as well as the recent exhibited 100 greater resistance to cefotaxime. The unanticipated development of synthetic glucokinase acti- potential for extending these efforts by combining site- vators. In fact, we have no way to reckon just how many directed-mutagenesis and chemical modification to improve central pathway activators and inhibitors remain to be the specificity of enzymes, especially those used by synthetic discovered. Moreover, although most enzymes are first organic chemists, should not be underestimated (Jones and discovered and isolated through the use of a well-defined Desantis, 1998) (see also Section 2.3: Active Site activity assay, one can never be absolutely certain that Diversification). a particular substrate is the physiologic substrate or that Chapter j 1 An Introduction to Enzyme Science 41
H3C O to define the structures of their active sites and regulatory sites Ph at atomic resolution. Consider the fact that the Protein Data O OH Bank (PDB) presently lists some 56,000 structures, with H3C O NH O nearly one-fourth of human origin. Some 49,000 structures CH3 were established by X-ray techniques, with 7,000 determined CH Ph 3 by NMR and fewer than 200 by EM. Also listed in the PDB O are ~2,100 nucleic acid structures, with ~1,200 from X-ray OH HO analysis, ~900 from NMR, and <20 from EM. For the nearly O 2,500 structures for protein-nucleic acid complexes, ~2,300 CH3 Ph O were determined by X-ray, ~150 by NMR, and <65 by EM. While the tally of 56,000 documents the impressive pace of Divinyl Adipate Thermolysin in (Salt-Activated) acquiring protein structures over the past half century, it gives tert-Amyl Alcohol a somewhat distorted view of how much we already know,
H3C O simply because the ligand-free and -bound structures and Ph mutant forms of certain proteins have been so intensively O OH H3C investigated that these proteins are disproportionately repre- O NH O sented in the PDB. Various hemoglobins, for example, CH3 account for ~1.6% of all PDB structures. Among the inten- CH Ph 3 sively studied enzymes are: lysozyme (2%), angiotensi- O O nogen-converting enzyme (~1.5%), RNase (~1.4%), the OH C O ribosome (~1.4%), trypsin (1.3%), chymotrypsin (~1%), O actin (1%), carbonic anhydrase (0.6%), adenylate kinase CH2=CH—O-C(=O)—(CH2)3—CH2 CH3 Ph O (~0.5%), and myosin (~0.4%). Collectively, the proteins lis- ted above represent one-eighth of all curated structures in the PDB! To fathom the degree to which the overall tally grossly Acetonitrile Lipase (solvent) Candida antarctica under-represents the proteome, one need only consider that human and mouse genomes each contain >20,000 protein-
H3C O encoding genes, with Drosophila at ~13,000, C. elegans at Ph ~17,000, Arabidopsis at ~28,000, rice at ~38,000, S. cer- O OH evisiae at ~6,000, and E. coli at ~5,000. In all, more than H3C O NH O 500,000 proteins would be needed to represent the proteomes CH3 of the 100 most frequently studied organisms and viruses. CH Ph 3 Even after allowing for the 10–15% that are fibrous and/or O O intrinsically disordered, upwards of 3–5 million different OH C O protein structures would be required to fully represent the O ligand-free and -bound states for the remaining globular HOOC—(CH2)3—CH2 CH3 Ph O proteins. An effort exclusively directed toward defining the structures of all human proteins would itself swell the current Scheme 1.12 PDB holdings by a factor of 5–10. Obviously, such a massive undertaking is presently infeasible and would require devel- opment of high-throughput robotic methods for efficiently other substrates are also metabolized. Many enzymes are expressing, purifying, crystallizing, and then structurally selective in their action toward substrates and are only analyzing such a vast array of protein structures. rarely exhibit absolute specificity. Nowhere is this state- To reveal telltale structural features underlying molec- ment truer than in the identification of the primary phos- ular recognition and substrate specificity, one need not phoryl-acceptor substrate for the numerous signal- possess an atomic-level structure for all enzymes within transducing protein kinases. An added issue is the a proteome. One may only need the structures of as few as phenomenon of ‘‘catalytic promiscuity’’ (see Section five to ten thousand more enzymes with numerous repre- 2.3.2), wherein a single enzyme operates by more than one sentatives from each reaction types found in the Enzyme catalytic mechanism, giving rise to multiple enzymatic Commission’s classification. Moreover, wider application activities. Catalytic promiscuity increases the likelihood of molecular docking with a suitably robust reference that we have unknowingly failed to identify many physi- library consisting of all known low-molecular-weight ologically important reactions. metabolites would develop criteria for reliably predicting Such concerns point the need for a far more comprehensive the most substrate specificity as well as the probable cata- X-ray and NMR investigation of many, many more enzymes lytic mechanism for those enzymes whose activities have 42 Enzyme Kinetics yet to be established experimentally. Computational occurring, disease-causing enzyme mutations, perhaps even approaches are also required to provide a means for effi- facilitating the design of custom-tailored therapeutic ciently re-surveying enzyme surfaces, again at atomic interventions. resolution, to find previously undiscovered crevices that Finally, by redesigning enzyme active sites to accom- serve as activator and inhibitor sites. Such work may also modate novel substrates, we face the welcome prospect that help us to understand how so many different proteins therapeutic enzymes may soon be re-fashioned in ways manage to co-exist within crowded compartments with allowing them to modify and/or detoxify natural and engaging in nonspecific aggregation. manmade toxins. Given the many millions of synthetic Although highly automated robotic acquisition of organic chemicals that have been prepared for commercial enzyme structures may provide us with a catalogue of high- and research purposes, the ability to re-jigger enzyme active resolution structural data, the value of such a treasure trove sites to catalyze novel reactions would increase the reme- of structural data would be underwhelming in the absence of dial potential during failures in chemical containment, commensurate advances in high-throughput biochemical especially if existing highly abundant enzymes can be characterization. What ultimately drives discovery science altered for such purposes. We may likewise anticipate the is the sense of intrigue and opportunity that researchers use of these synthetic enzymes in the conversion of pro- experience when they ponder the properties and complexity drugs (see Section 8.12.5) into their therapeutically active of an unsolved scientific problem. Without commensurate forms. growth in hypothesis-based, experimental enzymology, we would soon find, as put so well by Tennyson, that 1.6.6 We Need to Develop the Ability to ‘‘knowledge comes, but wisdom lingers.’’ Structural and Design Entirely New Biological Catalysts functional characterization of the entire human proteome would allow us to comprehend the full spectrum of ligand Given the trend toward minimizing the environmental binding interactions underlying enzyme catalysis and impact of chemical industries, greater emphasis must be control as well as to manage disease-causing enzyme placed on designing enzymes with new catalytic function. mutations through the design of new drugs and/or thera- Learning precisely how substrates approach and dock peutic interventions. within enzyme active sites should permit us to remodel To date, most molecular structure analyses stem from an active sites to create new catalysts. interest in a particular enzyme or its intriguing biochemical Shown in Fig. 1.9 is the enlightening and efficient multi- properties; even so, there is good reason to believe that we step strategy developed by Jiang et al. (2008) for the rational have not succeeded in identifying likely physiologically design of new enzymes, with their study focusing on the significant alternative substrates or all of the allosteric catalysis of retro-aldol reaction (Scheme 1.13). activators and inhibitors. There is thus an emerging recognition of the need for a more comprehensive investi- H3C gation of numerous active-site structures at atomic resolu- C CH2 O tion by X-ray and neutron crystallography. Such O CH CH a coordinated effort, which would focus on perhaps as few 3 as several thousand more enzymes representing every H3C Enzyme Commission reaction type, is likely to reveal telltale structural features that underlie substrate specificity. Enzyme Moreover, wider application of molecular docking with a suitably robust reference library consisting of all known H3C low-molecular-weight metabolites (i.e.,MW< 1–3 kDa) C CH3 would develop criteria for reliably predicting the most O O substrate specificity as well as the probable catalytic HC CH3 mechanism for those enzymes whose activities have yet to O be established experimentally. Efforts to perfect high- throughput computational approaches are also required to Scheme 1.13 provide a means for re-surveying all enzyme surfaces, again at atomic resolution, thereby fostering the development of In the first step of their computational enzyme design new ways to predict previously undiscovered activator and effort, Jiang et al. (2008) defined potential catalytic mech- inhibitor sites. Such efforts would fulfill a longstanding anisms for a retro-aldol-type reaction. Recall that this need to comprehend the fuller spectrum of ligand binding reaction proceeds in distinct stages (Scheme 1.14), each interactions responsible for cell, tissue, organ, and inter- involving acid/base catalysis by either amino acid side organ regulatory mechanisms. These same approaches can chains or water molecules (see also Fig. 2.27 describing be extended to the systematic investigation of naturally aldolase catalysis). Chapter j 1 An Introduction to Enzyme Science 43
H-bond H-bond :B :B O OH LYS :B OH OH NH OH H2O HN LYS NH 2 O O LYS O
:B H-bond H-bond O O :B H :B OH H O LYSNH2 + OH 2 + HN HN H2N O LYS LYS LYS
Scheme 1.14
Nucleophilic attack of an enzyme lysine on the sub- to have unfavorable catalytic geometry or to give rise to strate’s ketone group forms a carbinolamine intermediate, significant steric clashes. which upon eliminating water forms the imine/iminium After optimization of the composite TS rigid body species. Carbon–carbon bond cleavage is then triggered by orientation and the identities and conformations of the the deprotonation of the b-alcohol, with the iminium surrounding residues, a total of 72 designs with 8–20 intermediate acting as an electron sink. Finally, the enamine amino acid identity changes in 10 different scaffolds were tautomerizes to an imine, which is then hydrolyzed to selected for experimental characterization based on the release the covalently bound product and free the enzyme predicted TS binding energy, the extent of satisfaction of for another round of catalysis. Each elementary reaction in the catalytic geometry, the packing around the active such a multi-stage mechanism has its own transition state, lysine, and the consistency of side-chain conformation which must be stabilized by the enzyme. after side-chain repacking in the presence and absence of In the second step of the design process, Jiang et al. the TS model. cDNA’s encoding each design were con- (2008) identified known protein scaffolds that might structed and the proteins were expressed and purified from accommodate the designed TS ensemble described above. Escherichia coli, yielding soluble purified protein for 70 To account for the multi-step reaction pathway, they of 72 designs. designed a composite structure of acid/base groups that is Retro-aldolase activity was monitored via a fluores- simultaneously compatible with multiple transition states cence-based assay of product formation for each of the and anticipated reaction intermediates. In this effort, they designs. Their initial 12 designs used Motif I (Fig. 1.11B), generated design models using the four catalytic motifs which involves a charged side-chain (Lys-Asp-Lys)-medi- shown schematically in Fig. 1.10, which employ different ated proton transfer scheme resembling that for D-2- constellations of catalytic residues to facilitate carbinol- deoxyribose-5-phosphate aldolase. Of these designs, two amine formation and water elimination, carbon–carbon showed slow enaminone formation with 2,4-pentandione bond cleavage, and release of bound product. The authors (17), which is indicative of a nucleophilic lysine, but none emphasize that it is essential to consider a very large set of displayed retro-aldolase activity. Ten designs were made active-site possibilities, simply because the probability of based on Motif II, which is much simpler and involves accurately reconstructing a given three-dimensional active a single imine-forming lysine in a hydrophobic pocket, site in an input protein scaffold is extremely small. They similar to aldolase catalytic antibodies. Of these designs, generated such a set by simultaneously varying: (i) the one formed the enaminone, but none were catalytically internal degrees of freedom of the composite TS; (ii) the active. The third active site (Motif III) incorporates a His- orientation of the catalytic side chains with respect to Asp dyad as a general base to abstract a proton from the the composite TS, within ranges that are consistent with b-alcohol; of the fourteen designs tested, ten exhibited catalysis; and (iii) the conformations of the catalytic side stable enaminone formation, and eight had detectable retro- chains. This combinatorial matching resulted in a total of aldolase activity. In Motif IV, Jiang et al. (2008) experi- 181,555 distinct solutions for the placement of the mented with the explicit modeling of a water molecule, composite TS and the surrounding catalytic residues. positioned via side-chain hydrogen bonding groups, which The Rosetta Match algorithm rapidly eliminated most shuttles between stabilizing the carbinolamine and active-site possibilities in a given scaffold that are likely abstracting the proton from the hydroxyl. Of the thirty-six 44 Enzyme Kinetics
To evaluate the accuracy of the design models, Jiang et al. (2008) solved the structures of two of the designs by Compute TS for each step ˚ Select library of X-ray crystallography (Fig. 1.11). The 2.2-A resolution with optimally placed scaffold proteins protein functional groups structure (Panel D) showed that the designed catalytic residues Lys159, His233, and Asp53 superimpose well on the original design model, and the remainder of the active Combine to generate Identify pockets composite active site site is nearly identical to the design. The 1.9 A˚ resolution structure of the M48K variant of RA61 likewise reveals an active site very close to that of the design model, with only
Identify scaffold positions allowing construction of active site His46 and Trp178 in alternative rotamer conformations, perhaps resulting from the absence of substrate in the crystal structure (Panel E). Optimize composite TS and catalytic side-chain conformations What is so appealing about the work of Jiang et al. (2008) is that each proposed catalytic mechanism is treated as an Design neighboring positions for high affinity TS binding experimentally testable hypothesis through multiple inde- pendent design experiments. A candidate scaffold with its Optimize entire active site pendant catalytic groups can first be tested in silico by computer modeling protocols, then in vitro by kinetic Rank based on binding energy and catalytic geometry measurements, and finally in the crystal state by X-ray diffraction. The authors speculate that their computationally Experimentally characterize top ranking designs designed enzymes resemble primordial enzymes more than highly refined modern-day enzymes. In any case, Jiang et al. FIGURE 1.9 Computational design protocol for a multi-step enzyme- (2008) convincingly demonstrated that novel enzyme catalyzed reaction. Step-1: Generate ensembles of models of each of the activities can be designed from scratch through the use of key intermediates and transition states (TS) in the reaction pathway in the their systematic approach. context of a specific catalytic motif composed of protein functional groups. Step-2: Superimpose these models, based on the protein functional group positions, to create an initial composite active-site description. Step-3: Generate large ensembles of distinct 3D realization of these composite 1.6.7 We Need to Define the Efficient Routes active sites by simultaneously varying the degrees of freedom of the for Obtaining High Potency Enzyme composite TS, the orientation of the catalytic side chains relative to the composite TS, and the internal conformation of the catalytic side chains. Inhibitors as Drugs and Pesticides For each composite active site description, candidate catalytic sites are generated in an input scaffold set by Rosetta Match software (Zanghellini Enzyme inhibitors are by far the most effective drugs, et al., 2006). Briefly, each rotamer of each catalytic side-chain is placed at because an inhibitor’s effect on metabolism is magnified by each position within each scaffold, and the ensuing position of the composite the target enzyme’s catalytic efficiency. It’s also the case TS is recorded in the hash. After filling out the hash table, which is linear in that an enzyme’s specificity for its substrate(s) is often the numbers of scaffold positions and catalytic rotamers, the table is manifested in its interactions with inhibitors. searched for TS positions (termed ‘‘matches’’) that are compatible with all catalytic constraints; such positions are termed ‘‘matches.’’ Step-4: Opti- Hopkins and Groom (2002) concluded that only about mize the rigid body orientation of the composite TS and the internal coordi- 3,000 of the 30,000 genes in the human genome can be nates of the catalytic side chains for each match, reducing steric clashes classified as ‘‘disease-modifying genes.’’ The ever-expand- while maintaining the catalytic geometry within specified tolerances. The ing enterprise of developing the next cadre of billion-dollar remaining positions (not included in the minimal catalytic site description) drugs depends heavily on the discovery of new enzymes and surrounding the docked composite TS model are redesigned to optimize TS binding affinity by means of the standard Rosetta design methodology (Dan- inhibitors that may serve as drug targets and as lead mole- tas et al., 2003; Meiler and Baker, 2006). The rigid body orientation of the cules that guide drug design. Most drug discovery efforts composite TS, the side chain torsion angles, and (in some cases) the back- begin with the recognition that a compound shows promise bone torsion angles in the active site are refined via quasi-Newton optimiza- as an inhibitor of an enzyme of pharmacologic interest. Such tion. Step-5: Rank the resulting designs, based on the total binding energy to molecules, called lead compounds (or simply leads), must the composite TS and the satisfaction of the specified catalytic geometry. Step-6: Experimentally characterize the top-ranked designs. Figure and run the gauntlet of criteria for evaluating the promise of legend reproduced with minor modification from Jiang et al. (2008) with a new drug. Capitalizing on mode-of-action information, permission of the authors and the publisher. pharmacologists and medicinal chemists are perfecting strategies for developing novel drugs (Copeland, 2005). designs tested, twenty formed the enaminone and twenty- Combinatorial libraries of organic compounds are also three (with eleven distinct positions for the catalytic lysine) employed to identify leads based on the ability of randomly had significant retro-aldolase activity, with rate enhance- shaped molecules to fill cavities within an enzyme’s active ments up to four orders of magnitude over the uncatalyzed site. Genomics and proteomics are likewise being explored reaction. as new avenues for identifying lead molecules. Chapter j 1 An Introduction to Enzyme Science 45
FIGURE 1.10 Candidate motifs for catalysis of retro-aldol reaction mechanisms. Shown are active-site motifs with quantum mechanically optimized structures. Motif I, possessing two lysines positioned nearby each other to lower the pK a of the nucleophilic lysine, and a Lys- Asp dyad acting as the base to deprotonate the hydroxyl group. Motif II, with catalytic lysine buried in a hydrophobic environment
to lower its pKa, thereby increasing its nucleophilic character, and a tyrosine that can function as a general acid or base. HB, hydrogen bond. (Top right) Motif III, wherein the catalytic lysine (analogous to Motif II) is in a hydrophobic pocket to lower its pKa, and a His-Asp dyad serves as a general base similar to the catalytic unit commonly observed in the serine proteases. Motif IV, with the catalytic lysine is again positioned in a hydrophobic environment. Additionally, an explicitly modeled bound water molecule is placed, such that it forms a hydrogen bond with the carbinolamine hydroxyl during its formation, aids in the water elimination step, and deprotonates the b-alcohol at the C–C bond-breaking step. A hydrogen-bond donor/acceptor, such as Ser, Thr, or Tyr, is placed to position the water molecule in a tetrahedral geometry with the b-alcohol and the carbinolamine hydroxyl. The proton abstracting ability of the water molecule is enhanced by a second hydrogen bond with a base residue. We incorporated, where possible, additional hydrogen-bonding interactions to stabilize the carbinolamine hydroxyl group and an aromatic side chain to optimally pack along the planar aromatic moiety of the substrate. Figure and legend adapted from Jiang et al. (2008) are reproduced here with permission of the authors and the publisher.
The most reliable tools, by far, are the mechanistic quantity at a given time (Gillespie, 1992; Norris, 1997; insights obtained through kinetic analysis of enzyme action, van Kampen, 1992). A powerful justification for con- and such efforts will doubtlessly require advances in ducting single-molecule observations is the need to test enzyme science as well as structural biology, molecular whether individual members are indeed representative of mechanics, and physical biochemistry. A particularly the overall population of molecules (Xie and Trautman, fruitful approach is to infer the most likely transition-state 1998). Reaction trajectories can now be reliably deter- geometry through the determination of kinetic isotope mined for individual enzyme molecules that are physi- effects. These concepts and experimental strategies are cally isolated from each other by attachment to solid described in Chapters 8 and 9. surfaces or supramolecular structures, during confinement within a gel or polymer matrix, or as they operate cata- 1.6.8 We Need to Learn More About lytically and move freely within an extremely small volume element. As will become clear later in this In Singulo Enzyme Catalysis reference book, other breakthroughs in materials science Direct visualization of catalytic reaction cycles of an indi- and chemical physics have also spurred the development vidual enzyme molecule (hence the term in singulo)isat of single-molecule kinetics. long last feasible. Enzyme kinetic experiments have tradi- Enzyme chemists and statistical physicists are similarly tionally been carried out with large numbers of enzyme intrigued by the stochastics of enzyme catalysis and coop- molecules, and even 1-nL volume of 1 nM enzyme contains erativity (e.g., activity fluctuations, pausing, waiting-time nearly a million molecules. Advances in protein science, distributions, static disorder, fluctuating reactant concen- optics, fluorescence and solid-state electronics, however, trations, etc.). Such information affords the opportunity to make possible the direct observations of single enzyme compare individual and ensemble-averaged properties molecules. unambiguously, thereby bridging the microscopic and The ergodic hypothesis asserts that the time-average macroscopic worlds of chemistry. of a physical quantity along a time trajectory of an These concepts and the ever-expanding armamentarium individual member within a homogeneous ensemble is of experimental tools for testing them are explored more equivalent to the ensemble-averaged value of that fully in Chapter 12. 46 Enzyme Kinetics
FIGURE 1.11 Structures of computationally designed enzymes. A–C: Examples of design models for active site designs highlighting groups important for catalysis. The nucleophilic imine-forming lysine is in orange, the transition-state model is in yellow, the hydrogen-bonding groups are in light green, and the catalytic water is shown explicitly. The designed hydrophobic binding site for the aromatic portion of the TS model is indicated by the gray mesh. A: RA60 (catalytic motif IV, jelly-roll scaffold), wherein a designed hydrophobic pocket encloses the aromatic portion of the substrate and packs the aliphatic portion of the imine-forming Lys48. A designed hydrogen-bonding network positions the bridging water molecule and the composite TS. B: RA46 (catalytic motif IV, TIM-barrel scaffold), wherein Tyr-83 and Ser-210 position the bridging water molecule, thereby potentially facilitating required proton shuffling in active site IV. C: RA45 (catalytic motif IV, TIM-barrel scaffold). The bridging water is hydrogen-bonded by Ser-211 and Glu-233; replacing the Glu-233 with Thr decreases catalytic activity by a factor of three. D and E: Over- lay of design model (purple) on X-ray crystal structure (green). Designed amino acid side-chains are shown in stick representation, and the TS model in the design is shown in gray. D:The2.2A˚ crystal structure of the Ser-210-Ala variant of RA22 (catalytic motif III, TIM-barrel scaffold). The root mean square deviation (RMSD) for Ca atoms for the design model and its crystal structure is 0.62 A˚ , and the heavy-atom RMSD in the active-site is 1.10 A˚ . E:1.8A˚ crystal structure of Met-48-Lys variant of RA61 (catalytic motif IV, jelly-roll scaffold). Design-crystal structure Ca- atom RMSD is 0.46 A˚ , and heavy-atom RMSD is 0.8 A˚ . The small differences in the high-resolution details of packing around the active site are believed to arise from slight movements in some of the loops above the binding pocket and two rotamer changes in RA61 that may reflect the absence of a bound TS analogue in the crystal structure. Figure and legend adapted from Jaing et al. (2008) are reproduced here with permission of the authors and the publisher.
1.6.9 We Need to Develop Comprehensive (go to: http://www.ebi.ac.uk/thornton-srv/databases/MACiE/ Catalogs of Enzyme Mechanisms and to Use glossary.html) categorizes the reaction mechanisms of well-characterized enzymes in the Protein DataBase Such Information in Fashioning New (PDB). MACiE is a collaborative project between John Metabolic Pathways Mitchell’s Group at the Unilever Center for Molecular A promising development that should foster rational Information at Cambridge University and Janet Thorn- comparison of enzyme reaction mechanisms and perhaps ton’s research group at the European Bioinformatics even the design of new metabolic pathways is the Institute, located south of Cambridge. All curated MACiE database (Holliday et al., 2005, 2006). This mechanisms are taken from the primary literature by internet-accessible bioinformatics database standing for a suitably trained chemist and biochemist. Each enzyme Mechanism, Annotation and Classification in Enzymes is assigned an identifying number based on the Enzyme Chapter j 1 An Introduction to Enzyme Science 47
Commission system (go to: http://www.chem.qmul.ac.uk/ et al. (1998) described a methanol/O2 biofuel cell that iubmb/enzyme/). The MACiE database specifies: (a) reac- uses an NADþ-dependent dehydrogenase as catalysts and tion identifier; (b) overall reaction type; (c) atoms involved; exploits an electro-enzymatic method to regenerate (d) bonds involved; (e) bonds broken; (f) bonds made; (g) NADH at modest over-potentials. We may also surmise substrates, cofactors products, along with suitable Kegg that effective photo-electro-enzymatic methods will Ligand Database identifiers (go to: http://www.genome.jp/ likewise harness solar energy to create electrode over- ligand/); (h) groups transferred; (i) groups eliminated; potentials. (j) species reduced; and (k) species oxidized. Pointing to the overwhelming impact of human activity While admittedly more daunting, reaction stages are on Earth’s biosphere, futurists tell us that thermal pollu- annotated with respect to: (i) involved substrate, cofactor tion is unavoidable. Some suggest that the effects of and/or product; (ii) reaction centers; (iii) rate-determining global warming have been grossly underestimated, simply step? (iv) reversible step? (v) stage reaction type; (vi) because higher temperatures are suppressed by the buff- group(s) transferred; (vii) involved nucleophile; (viii) type ering effects of deep ocean currents; once these heat sinks of tautomerization; (ix) reaction type; (x) reaction attri- are loaded, unchecked ‘‘temperature creep’’ may mani- butes; (xi) bond cleaved; (xii) bond formed; (xiii) bond- festly become what may be regarded as human-generated order change; and (xiv) involved residues, whether heat. The only apparent counter-measure is inventive a nucleophile, charge stabilizer, spectator, etc. As described conservation, where new efficiencies must be realized by Holliday et al. (2005; 2006), the process of annotating through improved machine designs and/or where chemists the data contained within MACiE involves advanced devise better ways to transduce solar energy into chemi- methods to minimize erroneous data entry. Wherever cally stored energy. If chlorophyll is the answer,10 then possible, issues of semantics are resolved by reference to the one or more enzymes will likewise play a part. If calcium- IUPAC Gold Book (go to: http://goldbook.iupac.org/) as mediated depletion of CO2 is the answer, then the enzy- well as the MACiE dictionary (go to: http://www.mitchell. mology of biomineralization will enjoy mounting interest. ch.cam.ac.uk/macie/glossary.html). An added advantage And if bacterial fermentation is the answer, new pathways of MACiE is that it should become feasible to identify with enhanced enzymatic activities can be developed. U.S. known enzymes as best-case candidates for the generation Patent Number 5,000,000, for example, describes a genet- of novel catalysts via site-directed mutagenesis. Because ically engineered Escherichia coli that was transformed the overall reaction is treated as the composite of mecha- with alcohol dehydrogenase and pyruvate decarboxylase nistic steps, MACiE should eventually resolve short- genes from Zymomonas mobilis (Ingram, Conway and comings in the EC nomenclature of energase-class enzymes Alterthum, 1991). These genes are expressed at sufficient (Purich, 2001). levels to confer upon the resulting Escherichia coli As noted earlier, fully one-fifth of the gross national transformant an ability to produce ethanol fermentatively product of an industrialized country depends on catalysis. at 80–90% efficiency. This patent shows that bacterial Unfortunately, most synthetic catalysts exploit special enzymology is already playing a role in converting silage, properties of aluminum, chromium, manganese, nickel, corn syrup, and even biodegradable landfill refuse into platinum, palladium, ruthenium, etc., of which most are biofuels. inherently toxic as elemental metals or simple metal Another fertile approach, pursued by Synthetic Geno- oxides. Techniques that increase their effective surface mics, Inc., is the design of entirely novel metabolic path- area, such as atomic deposition on carbon or zeolites, ways using microorganisms that possess synthetic, or also increase their hydrolysis and undesired entry into stripped-down, genomes that are optimized to allow for the biosphere. Given the significance of catalysis in our single-purpose production of valuable substances, biofuels, everyday lives, it may be reasonably expected that etc. The goal is to modify the operating system of a cell to natural or ‘‘remanufactured’’ enzymes will play a major direct the synthesis of metabolic products with commercial role in efforts to develop a ‘‘Green Chemistry’’ that is value and improve those cellular properties essential for both efficient and ecologically sound. Because the large-scale commercial bioprocesses. cardinal features of enzymes are specificity and high turnover, and because enzymes are completely biocom- patible, enzyme science has much to offer in the devel- opment of catalysts affording high yields and low toxicity. For example, enzyme-catalyzed biofuel cells 10 The following simple calculation indicates that an artificial system with may soon offer an alternative to transition metal catalysts an efficiency comparable to photosynthesis would be a considerable source for power generation. They could, in principle, facilitate of renewable energy. In the U.S., ~2500 hours per year of sunlight reach an intensity of ~800 watts per square meter, meaning that one hectare oxidize alcohols at relatively low over-potential without (104 m2) receives ~2 1010 watt-hours of energy. If 50% of this solar 10 the production of detrimental carbon monoxide, and are energy could be harvested as H2, the energy output would be ~10 capable of operation at lower temperatures. Palmore watt-hours of energy. 48 Enzyme Kinetics
1.6.10 We Need to Understand How to dynamics of metabolic and physiological systems through Analyze the Kinetic Behavior of Discrete modeling and simulation that is cast in terms of the sensitivity or responsiveness of metabolic flux to input signals. Meta- Enzyme-Catalyzed Reactions as Well as bolic Control Analysis is introduced in Section 11.13. Metabolic Pathways in their Environment Whether such efforts successfully reproduce an enzy- Our knowledge about how individual enzymes actually me’s intracellular interactions is largely a matter for operate within cells is surprisingly meager. Systematic conjecture. Recognizing that the intracellular milieu may investigation of the intracellular kinetics of enzymes alter the kinetic behavior of enzymes, some investigators promises to enrich our understanding of discrete enzymatic have conducted in vitro kinetics using suspensions of per- processes as well as the flow of metabolic information that meabilized cells to eliminate barriers to intracellular action is encoded in the ligand binding kinetics and enzymic of an enzyme on substrate(s) supplied externally. The basic processes associated with signal transduction cascades. approach is to disrupt the peripheral membrane by multiple Enhanced understanding intracellular enzyme kinetics freeze-thaw cycles or by treatment with agents like digonin, promises to improve the ways in which drugs are designed filipin, Triton X-100, or Lubrol WX. The goal is to allow and used, including efforts to minimize harmful side- free access of low-molecular-weight substrates and meta- effects. bolic effectors to enzymes within treated cells without dis- While we might anticipate that the availability of high- lodging the enzyme of interest from its normal site and resolution microscopes and high-sensitivity color cameras certainly without loss of proteins from the permeabilized would facilitate studies of enzymatic kinetics within living cells. A good system is the yeast Saccharomyces cerevisiae, cells, little progress has been made on measuring enzyme the cell wall of which, even after peripheral membrane kinetics in situ. A major challenge is that spectral signals permeabilization, acts as a semipermeable barrier that from substrates and products for an individual enzyme retains intracellular proteins while permitting small mole- reactions are most often obliterated by spectral signals from cules to enter or leave (Chow and Palecek, 2004; Serrano, the many chromophores and fluorophores of numerous Ganceda and Ganceda, 1973). other metabolites. Consider, for example, the conversion of Students of muscle contraction long ago recognized the NADþ, which itself is virtually transparent at 340 nm, to power of cell permeabilization in managing the kinetics of the NADH, which strongly absorbs 340-nm light. The problem actomyosin (AM) mechanochemical cycle and in investi- is that NADþ and NADH are involved in so many oxido- gating the action of myosin light chain kinase in the contractile reductase reactions that cannot be uniquely associate an process. Both processes are ATP-dependent, and radioactive absorbance change with a particular enzyme-catalyzed ATP and/or photo-caged ATP (see Section 10.6.1) can be reaction. The only exception is the use of synthetic chro- supplied exogenously to suitably permeabilized muscle fibers. mogenic and fluorogenic alternative substrates in place of He et al. (1997), for example, measured the rate of inorganic their natural counterparts. Another challenge is that the phosphate (Pi) release, and hence overall ‘‘ATPase’’ activity of concentration of a particular enzyme may vary within rabbit psoas muscle in single, permeabilized muscle fibers that different subcellular regions. Living cells are also of irreg- were in rigor prior to laser flash photolysis of caged ATP in the 2þ ular thickness, making it impossible to apply Beer’s Law presence and absence of Ca . The rate of Pi release from $ $ (i.e., Abs ¼ 3cl). Likewise, fluorescence measurements are AM ADP Pi complex was likewise monitored, based on the confounded by light scattering, quenching, as well as inner- rise in the fluorescence signal of the Pi-sensitive probe formed filter effects. by covalent labeling of bacterial phosphate-binding protein Stable and radioactive isotopic tracers (see Chapters 4, 9, with the reporter group MDCC (see Section 4.5). Use of the and 11) are most often the best ways to analyze metabolic permeabilized muscle fiber approach also affords the oppor- flux, Ji, which is the net reaction rate (units ¼ DMolarity/Dt), tunity to pre-load myosin’s active sites with the non- through the ith step in a pathway. Except for the rare instances hydrolyzable analogues p(NH)ppA and p(CH2)ppA in order to where a gaseous metabolite (e.g., CO2, CO, H2,CH4,NO,or study hydrolysis-sensitive steps in the AM reaction cycle. N2) is assimilated or released, isotopic assays are rarely Because most of our knowledge of regulatory molecule continuous, and substrate consumption or product formation interactions is the result of painstaking in vitro reconsti- must be determined by mass spectrometry or liquid scintil- tution experiments using fractionated cell components, lation counting after a sample of cells is fixed, extracted, and Mura and Stadtman (1981) opted to use permeabilized separated. In a few cases, NMR can be used if the labeled bacterial cells to re-investigate the bicyclic protein species is present in sufficient quantities. The specialized field nucleotidylation cascade that was first discovered in known as Metabolic Control Analysis focuses on the Stadtman’s laboratory (see Section 11.11). This prototyp- measurement of metabolic fluxes to learn how integrated ical system for enzyme-catalyzed reversible covalent metabolic networks operate within its cellular context. In interconversion regulates the interconversion of dodeca- MCA, the researcher seeks to understand large-scale meric glutamine synthetase between its adenylylated Chapter j 1 An Introduction to Enzyme Science 49
(catalytically active) and unadenylylated (catalytically which requires the unprecedented stoichiometric cleavage inactive) forms (Adler, Purich and Stadtman, 1975; of the redox coenzyme to facilitate amide hydrolysis; Stadtman and Ginsburg, 1974). At high concentration, telomerases, which add stabilizing DNA repeats (e.g., ammonia suffices for glutamine in numerous amido-syn- TTAGGG in vertebrates) to chromosome ends; NAD+- thase reactions leading to such nitrogenous metabolites as dependent poly-ADPR polymerases (or PARPs), which histidine, N-acetyl glucosamine, and CTP. Earlier studies likewise modify chromosome stability; as well as a battery with isolated protein and enzyme components indicated of scores to hundreds of ATP hydrolysis-dependent, that the state of glutamine synthetase adenylylation chromatin-remodeling mechanoenzymes. Although geno- depended on indicators of ammonia availability: a-keto- mics provides an upper bound on the likely number of glutarate was found to be a signal for low ammonia unique enzymes, there is no reliable metric for quantifying availability, whereas glutamine was an indicator that the complexity of interactions among these catalysts and ammonia was plentiful. Mura and Stadtman (1981) found their many protein, nucleic acid, and low-molecular- that permeabilization of Escherichia coli cells resulted in weight metabolic effectors. In epigenetics, for example, complete retention of all protein components, presumably we are only beginning to glimpse how individual tissues the result of the bacterium’s Gram negative peptidogycan change during development, aging, and senescence to cell wall. They found that the state of glutamine synthe- modify the set-points for energy metabolism or how tase within permeabilized cells increased to a high state of epigenetic marks are maintained within an organism or adenylylation in the presence of ATP and glutamine, with how these epigenetic marks undergo multi-generational ~11 of the synthetase’s 12 subunits containing an O- transmission from parent to child, and to succeeding tyrosyl-AMP moiety. However, in the presence of a keto- generations. And when long-range gene regulation is glutarate, Pi, and ATP, the average number of O-tyrosyl- considered (e.g., the multiple gene-coordinating action of AMP residues decreased to ~2. Time-dependent changes the locus control region (LCR) for stage-specific expres- in the state of adenylylation that occur during incubations sion of hemoglobin genes within conceptus, fetus, of permeabilized cells in buffers containing these effectors neonate, and adult), the likely pivotal importance of ATP- can be arrested either by sonication in the cold or by the dependent mechanoenzymes in prying open appropriate addition of cetyl-trimethyl-ammonium bromide (to inac- highly compacted chromatin regions for active transcrip- tivate adenylyltransferase). Mura and Stadtman (1981) tion, while simultaneously limiting RNA polymerase thus established that Lubrol-permeabilized cells are access to those genes that must remain quiescent, cannot a reliable way to investigate the regulation of glutamine be overstated. synthetase adenylylation in situ. We may likewise anticipate that a strong collaboration Given the need for additional approaches for investi- among enzyme kineticists and molecular geneticists will gating intracellular enzyme kinetics, it should be possible also quicken the pace of discovery of novel chromosome to first permeabilize tightly adhered cells and then use an regulation at this dawning hour. Learning how and when over-layer of mineral oil to physically isolate each cell from various chromatin-remodeling mechanoenzymes find and the others. A chromogenic or fluorogenic substrate could interact with specific locations within the nucleus also then be micro-injected into the small volume of buffer promises to provide the opportunity to alter cell function surrounding a cell of interest, and the progress of the reac- and/or proliferation. Indeed, timely development of novel tion could then be sensed by absorption or fluorescence kinetic assays allowing one to probe the in situ action of spectroscopy. gene-regulating enzymes is of paramount importance. So also will be the rationale design of novel inhibitors that are based on systematic investigation of the kinetic and catalytic mechanisms of these enzymes. For example, 1.6.11 We Need to Develop Techniques systematic kinetic isotope effect studies on the NADþ- that Will Facilitate Investigation of dependent histone deacetylases and telomerases should Chromosomal Remodeling, Epigenetics, provide valuable clues about the transition-state structure and the Genetic Basis of Disease and Cell and its acid/base properties. As described in Sections 8.6.1, 8.12.4, and 9.6, such information is essential for Survival the design of high-affinity transition-state inhibitors. Few fields within the broad scope of the molecular life Ultimately, collaborations among enzyme kineticists and sciences are developing as rapidly as the fields of chro- molecular geneticists will also enlarge the tally of new mosomal remodeling and epigenetics. Recent thrusts in druggable target enzymes, thereby expanding the molecular genetics, for example, led to the discovery of opportunity to develop a wider spectrum of drugs and many novel chromatin-associated enzymes, including: therapeutic regimens that should improve the health, numerous DNA methylases, which are responsible for performance, and sustained vitality of plants and epigenetic marking; NAD+-dependent histone deacetylase, animals. 50 Enzyme Kinetics
1.6.12 We Need to Develop Effective direct therapeutic agents? To address this issue, we may Enzyme Preparations for Use in Direct first categorize therapeutic enzymes as those autologous enzymes – those that are already normally produced by Enzyme Therapy healthy subjects within a given species versus heterologous The speed and specificity of enzyme catalysis commends enzymes – those that originate in a different species. These direct enzyme therapy (i.e., the use of small quantities of categories may be further subdivided on the basis of certain enzymes as drugs to treat patients by modifying whether an enzyme normally operates within or outside metabolism and/or ridding cells of disease-producing the confines of a cell. Autologous extracellular enzyme metabolites or toxins). This strategy includes and goes replacement offers greatest promise, because these enzymes beyond enzyme replacement therapy, wherein a deficient, should exhibit limited immunogenicity, low toxicity, and inactive, or absent enzyme is replaced by gene therapy and, should already be well adapted to the inherently oxidizing less often, by infusion. The potential of direct enzyme environment outside cells. For those enzymes destined for therapy was first entertained over 50 years ago by Linus use in intracellular therapy, the researcher must overcome Pauling, who was the first to trace a molecular basis of the additional obstacle of delivering the enzyme to the a disease (sickle cell anemia) to a likely amino acid correct intracellular compartment as well as in a physio- substitution (later shown to be the Glu-to-Val mutation logically controlled concentration range. The use of foreign position-6 within the b-hemoglobin chain). For Pauling, the enzymes increases the likelihood that the host cells will objectives for direct enzyme therapy were deceptively exhibit apoptotic instability and that the enzyme may simple – identify a disease-causing enzyme defect or defi- undergo rapid turnover. By far, the greatest obstacles for the ciency and replace that enzyme with one having full cata- clinical efficacy of intracellular enzyme therapy will be lytic and/or regulatory capacity. specific or selective delivery of the enzyme to the proper Table 1.5 presents those cases in which direct enzyme cell/tissue target(s) and in the proper dosage. Except in therapy has been achieved or is nearing realization. Despite rare circumstances, expression vectors like adenovirus, many determined efforts, the successes are still far too few, adeno-associated virus, and lentivirus are rarely delivered inviting the question: What limits the use of enzymes as with adequate specificity, and surface expression of viral
TABLE 1.5 Selected Examples of Direct Enzyme Therapy
Adenosine deaminase Corrects adenosine deaminase-linked severe combined immune deficiency (ADA-SCID), by preventing accumulation of toxic metabolites that impair cellular and humoral immunity. Asparaginase and glutaminase Reduces the viability of asparagine- and glutamine-requiring tumor cells by hydrolyzing asparagine and glutamine. Collagenases Debrides skin lesions, including scar tissue, ulceration, burns, and infected blisters. Dermal RNases Inhibits RNase-sensitive organisms, when applied in conjunction with membrane-lyzing detergents. (Importantly, dermal RNase activity is not blocked by 5’-capping of mRNA.) DNase Treats chronic bronchitis by reducing bronchial mucous viscosity (a) by hydrolyzing DNA and (b) by forming high-affinity complex with actin monomers, thereby greatly reducing level of filamentous actin. a-Galactosidase A Treats a variety of clinical manifestations of Fabry’s disorder by reducing globotriaosylceramide that accumulates in different cell types. Glucocerebrosidase Treats Gaucher’s disease, which is by far the most common lysosomal storage disease. a-Glucosidase Ameliorates late-onset Type 2 Glycogen Storage (or Pompe) Disease, a progressive multi- system disease evoked by a deficiency of lysosomal acid a-glucosidase. Lactase Relieves gastrointestinal distress, flatulance, as well as skin lesions in 75% of all adults worldwide who metabolize lactose poorly. Lecithinized superoxide dismutase Ameliorates severe hypovolemia caused by increased blood vessel permeability following burns by using its lecithin group to bind securely to dermal membranes, thereby allowing destruction of surface superoxide. Lysozyme Prevents microbial overgrowth by lyzing cell walls of various human pathogens. Onconase (RNase) Treats cancer A by triggering apoptosis as a consequence of messenger RNA and micro RNA degradation. Oxalate decarboxylase Reduces renal calcium oxalate monohydrate stone formation by decomposing dietary oxalate. Phenylalanine ammonia lyase Treats phenylketonuria by reducing serum levels of phenylalanine, which is converted to toxic phenylpyruvate. Proteases Treats bacterial infection by hydrolyzing pathogen cell walls and microbial biofilms. Some preparations also reduce HIV infection. Chapter j 1 An Introduction to Enzyme Science 51 antigens raises the specter of cellular immunity and FURTHER READING apoptosis. A highly efficient means for incorporating enzymes and other proteins into cells is afforded by add- Abeles, R. H., Frey, P. A., & Jencks, W. P. (1992). Biochemistry. Boston: ing the membrane-penetrating (or penetratin) sequences Jones and Bartlett. pp. 838. RQIKIWFQNRRMKWKK and RRRQRRKKR, found Altman, S. (1989). Ribonuclease P: an Enzyme with a Catalytic RNA respectively within Drosophila antennapedia and HIV-TAT Subunit. Adv. Enzymol., 62,1. Cech, T. R. (Ed.). (1993). The RNA World. Cold Spring Harbor, New York: proteins, to the primary sequence of potential therapeutic Cold Spring Harbor Press. pp. 239. enzymes. These sequences allow rapid and direct incorpo- Copeland, R. A. (2005). Evaluation of Enzyme Inhibitors in Drug ration of proteins into the cytoplasm of all cells tested to Discovery: A Guide for Medicinal Chemists and Pharmacologists. date. Even so, delivery to the proper cell target remains Hoboken: Wiley-Interscience. pp. 271. problematical. Whether delivered by means of viral vectors Frey, P. A., & Hegeman, A. D. (2006). Enzymatic Reaction Mechanisms. or as penetratin-containing fusion enzyme, the elusive goal New York: Oxford University Press. pp. 768. of maintaining enzyme dosage within a narrow well Guerrier-Takada, C., Gardiner, K., Maresh, T., Pace, N., & Altman, S. controlled range represents the Holy Grail for direct enzyme (1983). The RNA Moiety of Ribonuclease P is the Catalytic Subunit therapy. of the Enzyme. Cell, 35, 849. As potential therapeutic enzymes are identified and Hammes, G. G. (2002). Multiple Conformational Changes in Enzyme developed, we can be reasonably certain that site-directed Catalysis. Biochemistry, 41, 8221. Haldane, J. B. S. (1930). Enzymes. London: Longmans-Green. mutagenesis and chemical modification (e.g., conjugation to Jencks, W. P. (1969). Catalysis in Chemistry and Enzymology. San Fran- polyethylene glycol for reduced immunogenicity or to cisco: McGraw-Hill. lecithin or by recombinant methods to introduce CAAX- Metzler, D. E. (2004). Biochemistry: The Chemical Reactions of Biolog- type acylation sequences for enhanced membrane docking) ical Systems. New York: Academic Press. will be essential tools for adapting these enzymes for clin- Purich, D. L. (2001). Enzyme Catalysis: A New Definition Accounting for ical use. As will be discussed in Section 7.15.4, every site- Non-covalent Substrate- and Product-like States,. Trends in Biochem. directed enzyme mutant must also be treated as though it is Sci., 26, 417. an entirely new enzyme, each potentially with its unique Purich, D. L., & Allison, R. D. (2002). The Enzyme Reference. New York: physical and chemical properties. The same may be said for Academic Press. chemical modified enzymes. Such statements point to the Russell, C. A. (2004). Advances in Organic Chemistry Over the Last 100 need for substantial kinetic characterization of these Years. Annu. Rep. Prog. Chem., Sect. B., 100,3. Sinnott, M. (Ed.). (1998). Comprehensive Biological Catalysis: A Mech- modified enzymes to verify their likely effectiveness. Also anistic Reference, vols I–IV. San Diego: Academic Press. required are appropriate kinetic tests of the efficiency of Voet, D., & Voet, J. G. (2003). Biochemistry (3rd ed.). New York: J. Wiley. enzyme dissolution and dispersion when formulated Zaug, A. J., & Cech, T. R. (1986). The Intervening Sequence RNA of enzymes are introduced into blood or model cell types as Tetrahymena is an Enzyme. Science, 231, 470. well as kinetic measurements of enzyme turnover.