CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS
coelenterazine
The activity of an enzyme is responsible for the glow of the luminescent jellyfish at left. The enzyme aequorin catalyzes the oxidation of a compound by oxygen in the
presence of calcium to release CO2 and light CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS Introduction . Enzymes are large biological molecules that catalyze chemical transformations . The molecule also mediates the transformation of one form of energy into another . 25% of the genes in the human genome encode enzymes . Catalytic power and specificity – important characteristics . Active site – the place where enzymatic reactions take place CHAPTER 8. ENZYMES: BASIC CONCEPTS AND KINETICS Introduction . Nearly all known enzymes are proteins • RNA can function as a catalyst . Proximity effect – enzymes bring substrates closer together in an optimal orientation . They catalyze reactions by stabilizing transition states 8.1 CATALYTIC POWER AND SPECIFICITY Rate enhancement by enzymes . Enzymes accelerate reactions by factors of as much as a million or more 8.1 CATALYTIC POWER AND SPECIFICITY Substrate specificity . A substrate is a molecule upon which an enzyme acts . Some proteases cleaves specific amide bonds in a peptide . The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme . This precision is a result of the intricate 3D structure of the enzyme protein
Fig 8.1 Enzyme specificity. (A) Trypsin cleaves on the carboxyl side of arginine and lysine residues, whereas (B) thrombin cleaves Arg-Gly bonds in particular sequences only 8.1 CATALYTIC POWER AND SPECIFICITY Cofactors . The catalytic activity of enzymes depends on the presence of small molecules termed cofactors . Two groups of cofactors: metals and small organic molecules (coenzymes) . Tightly bound coenzymes are called prosthetic groups 8.1 CATALYTIC POWER AND SPECIFICITY Energy transformation . Enzymes can transform energy from one form into another . Photosynthesis – conversion of light E into chemical-bond E . Cellular respiration – conversion of the E in food into an ion gradient and the E in ATP . Myosin converts the E of ATP into the mechanical E of contracting muscles . Pumps in the membranes use the E of ATP to transport molecules and ions across the membrane . Enzymes play fundamental roles in these processes 8.2 FREE ENERGY Free energy change . The free energy change (ΔG) of a reaction tells us if the reaction can take place spontaneously • A reaction can take place spontaneously only if ΔG<0 • A system is at equilibrium if ΔG=0 • A reaction cannot take place spontaneously if ΔG>0 • ΔG depends only on the free energy of the products minus the free energy of the reactants; independent of the path of the transformation • The ΔG provides no information about the rate of a reaction • The reaction rate depends on the free energy of activation (ΔG‡) 8.2 FREE ENERGY Free energy and equilibrium . Consider a reaction A + B ← → C + D [C][D] G = G + RT ln G: standard free energy change [A][B] . If we define G' as the standard free energy change at pH=7 [C][D] G = G' + RT ln [A][B] . At equilibrium, [C][D] G' = −RT ln = −RT lnK' [A][B] eq
−G'/RT K'eq = e 8.2 FREE ENERGY Free energy and reaction rate . Enzymes alter only the reaction rate and not the reaction equilibrium . Enzymes accelerate the attainment of equilibria but do not shift their positions . The equilibrium position is a function only of the ΔG between reactants and products
Fig 8.2 Enzymes accelerate the reaction rate. The same equilibrium point is reached but much more quickly in the presence of an enzyme. 8.3 THE TRANSITION STATE Enzyme and activation energy . A chemical reaction of substrate S to form product P goes through a transition state, X‡ S → X‡ → P
. The difference in free energy between the transition state and the substrate is called the activation energy, ΔG‡
‡ ‡ ΔG = GX − GS
Fig 8.3 Enzymes decrease the activation energy. Enzymes accelerate reactions by decreasing ΔG‡, the free energy of activation. 8.3 THE TRANSITION STATE Enzyme-substrate complex . Much of the catalytic power of enzymes comes from their bringing substrates together in favorable orientations . The enzyme-substrate complex can be seen by the following results: 1. The fact that an enzyme- catalyzed reaction has a maximal velocity suggests the formation of a discrete ES complex
Fig 8.4 Reaction velocity vs substrate concentration in an enzyme-catalyzed reaction. 8.3 THE TRANSITION STATE Enzyme-substrate complex . The enzyme-substrate complex can be seen by the following results: 2. X-ray crystal structure of an enzyme-substrate complex 3. Spectroscopic studies
Fig 8.5 Structure of cytochrome P450 complexed with its substrate camphor. 8.3 THE TRANSITION STATE The active sites of enzymes . The active site of an enzyme is the region that binds the substrates . The interaction of the enzyme and substrate at the active site promotes the formation of the transition state and lowers the ΔG‡ of the reaction . There are some common features in the active sites of enzymes 1. The active site is a three dimensional cleft formed by groups that come from different parts of the amino acid sequence
Fig 8.6 Active sites may include distant residues. 8.3 THE TRANSITION STATE The active sites of enzymes . There are some common features in the active sites of enzymes 2. The active site takes up a small part of the total volume of an enzyme – the rest parts serve as a scaffold and regulatory sites 3. Active sites are unique microenvironments – optimized for substrate binding and catalysis 4. Substrates are bound to enzymes by multiple weak attractions – electrostatic, H bonds, and van der Waals forces 8.3 THE TRANSITION STATE The active sites of enzymes . There are some common features in the active sites of enzymes 5. The specificity of binding depends on the precisely defined arrangement of atoms in an active site
Fig 8.8 Lock-and-key model of enzyme- Fig 8.9 Induced-fit model of enzyme- substrate binding. Proposed by Emil Fischer substrate binding. Proposed by Daniel in 1890. Koshland in 1958. 8.3 THE TRANSITION STATE Substrate binding energy . Free energy is released by the formation of a large number of weak interactions between a complementary enzyme and its substrate . The full complement of such interactions is formed only when the substrate is converted into the transition state . The maximal binding energy is released when the enzyme facilitates the formation of the transition state – lowering activation energy 8.4 THE MICHAELIS-MENTEN MODEL First-order reactions
. A first-order reaction is one whose rate depends on the concentration of a single reactant raised to the first power . Consider a reaction: A → P . We can express the rate law
V = k[A] 8.4 THE MICHAELIS-MENTEN MODEL Second-order reactions
. A second-order reaction is one whose rate depends on the concentration of two different reactants, each raised to the first power . Consider a reaction: 2A → P or A + B → P . We can express the rate law V = k[A]2 or V = k[A][B] . pseudo-first order reactions ([A] << [B]) – no dependence of the rate on [B] 8.4 THE MICHAELIS-MENTEN MODEL Initial velocity and substrate concentration . The simplest way to investigate the reaction rate is to follow the increase in reaction product as a function of time
. Initial velocity (V0) is measured in different concentration ([S]) of
the substrate and V0 is plotted as a function of [S]; typically saturation curve is observed in high [S]
Fig 8.10 Determining the relation between initial velocity and substrate concentration. 8.4 THE MICHAELIS-MENTEN MODEL The Michaelis-Menten equation . Conditions • The concentration of the enzyme is negligible • The initial rate of formation is measured
Fig 8.11 Michaelis-Menten kinetics. 8.4 THE MICHAELIS-MENTEN MODEL Steady State Kinetics . The steady state is the situation in which the value of a particular quantity is constant • Its rate of formation is balanced by its rate of destruction • The population of a country in a steady state • In enzyme kinetics, the concept is applied to the concentration of enzyme-bound intermediates Pre-steady state Once the intermediates reach their steady state concentrations, the reaction rate changes relatively slowly with time. During this steady state, the rates of enzymatic reactions are traditionally measured 8.4 THE MICHAELIS-MENTEN MODEL The Michaelis-Menten equation
Rate of formation of ES
Rate of breakdown of ES
If we use the steady state approximation for [ES] Rate of formation of ES = Rate of breakdown of ES
The equation is rearranged and the ratio of the rate constants is ····· 1
defined as KM. 8.4 THE MICHAELIS-MENTEN MODEL The Michaelis-Menten equation
Because we measure initial velocities, [S] is equal to the initial [S]. Because we cannot measure [E], [E] is needed to be expressed by measurable quantities. (Enzyme exists in the two forms)
····· 2
By using Eq. 1 and 2, [ES] is expressed as: 8.4 THE MICHAELIS-MENTEN MODEL The Michaelis-Menten equation
V0 becomes the maximum when all enzymes are bound to S. ([ES] = [E]0) . Note th 1. When [S]>>KM, V0 = Vmax (0 order) When [S]< 2. When [S] = KM, V0 = Vmax/2 Therefore, KM is equal to the substrate concentration at which the reaction rate is half its maximal value 8.4 THE MICHAELIS-MENTEN MODEL KM of aldehyde dehydrogenase . Ethanol-sensitive persons exhibit facial flushing and rapid heart rate (tachycardia) after ingesting alcohol . In the liver, alcohol dehydrogenase converts ethanol into acetaldehyde . The acetaldehyde is the cause of the symptoms and processed to acetate by aldehyde dehydrogenase . The sensitive persons have the E478K mutation on one of two aldehyde dehydrogenases which causes low catalytic activity (by increasing KM) and low stability 8.4 THE MICHAELIS-MENTEN MODEL How to determine KM and Vmax . What you need to measure is: • The initial velocities at different substrate concentrations 8.4 THE MICHAELIS-MENTEN MODEL How to determine KM and Vmax . Lineweaver-Burk Plot Fig 8.12 A double-reciprocal or Lineweaver-Burk plot. 8.4 THE MICHAELIS-MENTEN MODEL The Significance of KM and kcat . The meaning of KM -1 -7 • For most enzymes, KM lies between 10 and 10 M • [S] at which half the active sites are filled; a measure of [S] required for significant catalysis to take place • KM is equal to the dissociation constant of the ES complex if k2 is much smaller than k-1 8.4 THE MICHAELIS-MENTEN MODEL The Significance of KM and kcat . The meaning of kcat • The turn over number of an enzyme is the number of substrate molecules converted into products by an enzyme molecule in a unit time when the enzyme is fully saturated with S • It is equal to the rate constant k2 , which is also called kcat • kcat is simply the first-order rate constant for the conversion of the ES complex to the EP complex 8.4 THE MICHAELIS-MENTEN MODEL The Significance of KM and kcat . Most enzymes are not normally saturated with substrate • Under physiological conditions, the [S]/KM ratio is typically between 0.01 and 1.0 . When [S] << KM, the value of kcat/KM can be used as a measure of catalytic efficiency • The value takes into account both the rate of catalysis with a particular substrate and the strength of the enzyme-substrate interaction • We can compare an enzyme’s preference for different substrates. (see the table in the next slide) 8.4 THE MICHAELIS-MENTEN MODEL The Significance of KM and kcat . The value of kcat/KM (< k1) can not be faster than the diffusion- controlled encounter of an enzyme and its substrate (<1010 s-1M-1) 8.4 THE MICHAELIS-MENTEN MODEL The Significance of KM and kcat 8.4 THE MICHAELIS-MENTEN MODEL Multiple substrates . Most reactions in biological systems start with multiple substrates and yield multiple products A + B ← → P + Q . Multiple substrate reactions can be divided into two classes: • Sequential reactions • Double-displacement reactions 8.4 THE MICHAELIS-MENTEN MODEL Multiple substrates . Sequential reactions • In sequential reactions, all substrates must bind to the enzyme before any product is released • The order of the addition of substrates and the release of products can be ordered or random • Many enzymes using NADH as a substrate exhibit the ordered sequential mechanism Lactate dehydrogenase 8.4 THE MICHAELIS-MENTEN MODEL Multiple substrates . Sequential reactions • In the random sequential mechanism, the order of the addition of substrates and the release of products is random • In the following reaction, either creatine or ATP may bind first, and either phosphocreatine or ADP may be released first Creatine kinase 8.4 THE MICHAELIS-MENTEN MODEL Multiple substrates . Double-displacement (ping-pong) reactions • One or more products are released before all substrates bind the enzyme • The enzyme is temporarily modified – a substituted enzyme intermediate Aspartate aminotransferase 8.4 THE MICHAELIS-MENTEN MODEL Allosteric enzymes . The Michaelis-Menten model is simple and broadly applicable . The model has greatly improved our understanding of enzyme kinetics . However, the model cannot account for the kinetic properties of many enzymes . Allosteric enzymes do not obey Michaelis-Menten kinetics • They display a sigmoidal dependence of reaction velocity on substrate concentration Fig 8.13 Kinetics for an allosteric enzyme. 8.5 ENZYME INHIBITION Enzyme inhibitors . The activity of many enzymes can be modulated by the binding of specific small molecules and ions . This means of modulating enzyme activity serves as a major control mechanism in biological systems . Many drugs and toxic agents act by inhibiting enzymes . Inhibition can be a source of insight into the mechanism of enzyme action • Specific inhibitors can often be used to identify residues critical for catalysis . Information for enzyme action helps design enzyme inhibitors – transition state analogs 8.5 ENZYME INHIBITION Enzyme inhibitors . Enzyme inhibition can be either irreversible or reversible . Irreversible inhibitors • They dissociate very slowly from its target enzyme • They tightly bind the enzyme (covalently or noncovalently) • Penicillin and aspirin 8.5 ENZYME INHIBITION Enzyme inhibitors . Reversible inhibitors – characterized by reversible binding to enzyme and a rapid dissociation of the enzyme-inhibitor complex • Competitive inhibitors • Uncompetitive inhibitors • Noncompetitive inhibitors Fig 8.14 Distinction between reversible inhibitors. 8.5 ENZYME INHIBITION Enzyme inhibitors . Competitive inhibitors • They often resemble the substrate and bind to the active site of the enzyme • In the presence of a competitive inhibitor, the substrate is prevented from binding to the same active site • A competitive inhibitor diminishes the rate of catalysis by reducing the proportion of enzyme molecules bound to a substrate 8.5 ENZYME INHIBITION Enzyme inhibitors . Competitive inhibitors • The maximum velocity (Vmax) of the reaction is unchanged • The apparent affinity of the substrate to the binding site is decreased • Any given competitive inhibitor concentration can be overcome by increasing the substrate concentration • They are commonly used as drugs (ibuprofen and statins) 8.5 ENZYME INHIBITION Enzyme inhibitors . Competitive Inhibitors Fig 8.16 Kinetics of a competitive inhibitor. 8.5 ENZYME INHIBITION Enzyme inhibitors . Uncompetitive inhibitors • The inhibitors bind only to the complex formed between the enzyme and the substrate (the E-S complex). • Uncompetitive inhibition cannot be overcome by the addition of The ESI complex does not go on to form any product more substrate • The broad-spectrum herbicide glyphosate (Roundup) is an uncompetitive inhibitor Glyphosate (Roundup) 8.5 ENZYME INHIBITION Enzyme inhibitors . Uncompetitive inhibitors • Increases the enzyme's apparent affinity for the substrate through Le Chatelier's principle (KM is lowered) • Decreases the maximum enzyme activity (Vmax) ([ES] decreases) Fig 8.17 Kinetics of an uncompetitive inhibitor. 8.5 ENZYME INHIBITION Enzyme inhibitors . Noncompetitive inhibitors • The inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites • When both the substrate and the inhibitor are bound, the enzyme- substrate-inhibitor complex The ESI complex does not go cannot form product on to form any product • The inhibition cannot be overcome by increasing the substrate concentration 8.5 ENZYME INHIBITION Enzyme inhibitors . Noncompetitive inhibitors • Decreases the maximum enzyme activity (Vmax) (functional [E] decreases) • KM is unchanged Fig 8.18 Kinetics of a noncompetitive inhibitor. 8.5 ENZYME INHIBITION Enzyme inhibitors . Noncompetitive inhibitors • Decreases the maximum enzyme activity (Vmax) (functional [E] decreases) • KM is unchanged Figure Illustration of a possible mechanism of non-competitive inhibition. 8.5 ENZYME INHIBITION Enzyme inhibitors . Double-reciprocal plots of reversible Inhibition • Useful for distinguishing between competitive, uncompetitive, and noncompetititve No inhibitor Competitive inhibitor 8.5 ENZYME INHIBITION Enzyme inhibitors No inhibitor Uncompetitive inhibitor Noncompetitive inhibitor 8.5 ENZYME INHIBITION How to get Ki value . Dixon plot – for competitive inhibitors 180 1/ 0 150 120 [ S ] = 2 0 0 M 90 60 [ S ] = 4 0 0 M K = 3 8 0 n M i 30 - 9 0 0 - 6 0 0 - 3 0 0 0 300 600 900 [ I ] , n M 8.5 ENZYME INHIBITION Irreversible inhibitors . Irreversible inhibitors can be used as an alternative to X-ray crystallography to study mechanisms of enzyme action . Irreversible inhibitors can be divided into three categories • Group-specific reagents • Reactive substrate analogs (affinity labels) • Suicide inhibitors 8.5 ENZYME INHIBITION Irreversible inhibitors . Group-specific reagents • Group-specific reagents react with specific side chains of amino acids • DIPF modifies only 1 of 28 serine residues in chymotrypsin • The result shows that this Ser is especially reactive • Better specificity is required Diisopropylphosphofluoridate Figure 8.22 Enzyme inhibition by DIPF, a (DIPF): a potent nerve gas group-specific reagent. 8.5 ENZYME INHIBITION Irreversible inhibitors . Reactive substrate analogs (affinity labels) • The molecules are structurally similar to the substrate • They covalently bind to active-site residues • More specific than group-specific reagents Figure 8.23,24 Affinity labeling. 8.5 ENZYME INHIBITION Irreversible inhibitors . Mechanism-based inactivators (suicide inhibitors) • These inhibitors are modified substrates generating a chemical intermediate that inactivates the enzyme through covalent modification Figure 8.25 Mechanism-based (suicide) inhibition. Monoamine oxidase is an important enzyme for neurotransmittor synthesis. The enzyme requires the cofactor FAD (flavin adenine dinucleotide) 8.5 ENZYME INHIBITION Irreversible inhibitors . Mechanism-based inactivators (suicide inhibitors) • Monoamine oxidase deaminates neurotransmitters such as dopamine and serotonin, lowering their levels in the brain • Parkinson disease and depression are associated with low levels of dopamine and serotonin, respectively • (-)Deprenyl is a suicide inhibitor of monoamine oxidase dopamine serotonine 8.5 ENZYME INHIBITION Transition-state analogs . In 1948, Linus Pauling proposed: • Compounds resembling the transition state of a catalyzed reaction should be very potent inhibitors • These compounds are called transition-state analogs . The inhibitor pyrrole 2-carboxylate binds to the racemase 160 times as tightly as does proline . Highly potent and specific inhibitors of enzyme can be produced by synthesizing transition state analogs Figure 8.26 Inhibition by transition-state analogs. The isomerization of L-proline to D-proline by proline racemase. 8.5 ENZYME INHIBITION Catalytic antibodies . Catalytic antibodies can be produced by using transition-state analogs as antigens Figure 8.27 N-Methylmesoporphyrin is a transition-state analog used to generate catalytic antibodies. By generating catalytic antibodies from the molecule, 2500-fold faster metallation was achieved. . The power of transition-state analogs: • Sources of insight into catalytic mechanisms • Potent and specific inhibitors of enzymes • Can be used as immunogens to generate a wide range of novel catalysts 8.5 ENZYME INHIBITION Catalytic antibodies Peter G. Schultz One of the pioneers of catalytic antibodies 8.5 ENZYME INHIBITION Inhibition of Penicillin . Penicillin is the first antibiotic discovered and -lactam containing suicide inhibitor Figure 8.28 The reactive site of penicillin is the peptide bond of its β-lactam ring. (A) Structural formula of penicillin. (B) Representation of benzylpenicillin. 8.5 ENZYME INHIBITION Inhibition of penicillin . Bacterial cell wall is made up of a macromolecule, called a sugars peptidoglycan . The peptidoglycan consists tetrapeptides of linear polysaccharide pentaglycine chains that are crosslinked bridges by short peptides . The crosslinking confers mechanical support and prevents bacteria from bursting Figure 8.29 Schematic representation of the peptidoglycan in Staphylococcus aureus. 8.5 ENZYME INHIBITION Inhibition of penicillin . Glycopeptide transpeptidase catalyzes the formation of the crosslinks . The crosslinks use D-amino acids Figure 8.30 Formation of cross-links in S. aureus peptidoglycan 8.5 ENZYME INHIBITION Inhibition of penicillin . Glycopeptide transpeptidase forms an acyl intermediate with the D-Ala of the D-Ala-D-Ala peptide . This intermediate then reacts with the amino group of the terminal glycine in another peptide to form the crosslink Figure 8.31 Transpeptidation reaction. 8.5 ENZYME INHIBITION Inhibition of penicillin . Penicillin resembles the D-Ala-D-Ala moiety of the normal substrate . The penicilloyl-enzyme does not react further