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 À40 kJ/mol) under the full range of conditions experienced by that and acetyl-phosphate (DGhydrolysis z À60 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 MgATP2À, 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 À1 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 À1 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 10À15 sÀ1, 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 bÀg P–O bond in E þ P, where ES1,ES2,EX,EP1, and EP2 are various enzyme-bound MgATP2À is around 10À4 to 10À6 sÀ1, 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 2À group transfer reactions (e.g., MgATP þ Acceptor # Phosphoryl À 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)