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Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 5 Properties of Enzymes Prentice Hall c2002 Chapter 5 Copyright © 2006 Pearson Prentice 1 Hall, Inc. Chapter 5 - Properties of Enzymes • Catalyst - speeds up attainment of reaction equilibrium • Enzymatic reactions - 103 to 1017 faster than the corresponding uncatalyzed reactions • Substrates - highly specific reactants for enzymes Prentice Hall c2002 Chapter 5 2 Properties of enzymes (continued) • Stereospecificity - many enzymes act upon only one stereoisomer of a substrate • Reaction specificity - enzyme product yields are essentially 100% (there is no formation of wasteful byproducts) • Active site - where enzyme reactions take place Prentice Hall c2002 Chapter 5 3 Prentice Hall c2002 Chapter 5 4 5.1 The Six Classes of Enzymes 1. Oxidoreductases (dehydrogenases) • Catalyze oxidation-reduction reactions Prentice Hall c2002 Chapter 5 5 2. Transferases • Catalyze group transfer reactions Prentice Hall c2002 Chapter 5 6 3. Hydrolases • Catalyze hydrolysis reactions where water is the acceptor of the transferred group Prentice Hall c2002 Chapter 5 7 4. Lyases • Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination (Synthases catalyze the addition to a double bond, the reverse reaction of a lyase) Prentice Hall c2002 Chapter 5 8 5. Isomerases • Catalyze isomerization reactions Prentice Hall c2002 Chapter 5 9 6. Ligases (synthetases) • Catalyze ligation, or joining of two substrates • Require chemical energy (e.g. ATP) Prentice Hall c2002 Chapter 5 10 5.2 Kinetic Experiments Reveal Enzyme Properties A. Chemical Kinetics • Experiments examine the amount of product (P) formed per unit of time (D[P] / Dt) • Velocity (v) - the rate of a reaction (varies with reactant concentration) • Rate constant (k) - indicates the speed or efficiency of a reaction Prentice Hall c2002 Chapter 5 11 First order rate equation • Rate for nonenzymatic conversion of substrate (S) to product (P) in a first order reaction: (k is expressed in reciprocal time units (s-1)) D[P] / Dt = v = k[S] Prentice Hall c2002 Chapter 5 12 Second order reaction • For reactions: S1 + S2 P1 + P2 • Rate is determined by the concentration of both substrates • Rate equation: v = k[S1]1[S2]1 Prentice Hall c2002 Chapter 5 13 Pseudo first order reaction • If the concentration of one reactant is so high that it remains essentially constant, reaction becomes zero order with respect to that reactant • Overall reaction is then pseudo first-order v = k[S1]1[S2]0 = k’[S1]1 Prentice Hall c2002 Chapter 5 14 B. Enzyme Kinetics • Enzyme-substrate complex (ES) - complex formed when specific substrates fit into the enzyme active site E + S ES E+P • When [S] >> [E], every enzyme binds a molecule of substrate (enzyme is saturated with substrate) • Under these conditions the rate depends only upon [E], and the reaction is pseudo-first order Prentice Hall c2002 Chapter 5 15 Fig 5.1 Effect of enzyme concentration [E] on velocity (v) • Fixed, saturating [S] • Pseudo-first order enzyme-catalyzed reaction Prentice Hall c2002 Chapter 5 16 Initial velocity (vo) • Velocity at the beginning of an enzyme-catalyzed reaction is vo (initial velocity) • k1 and k-1 represent rapid noncovalent association /dissociation of substrate from enzyme active site • k2 = rate constant for formation of product from ES E+S Prentice Hall c2002 k1 k-1 ES Chapter 5 k2 E+P 17 Fig 5.2 Progress curve for an enzyme-catalyzed reaction • The initial velocity (vo) is the slope of the initial linear portion of the curve • Rate of the reaction doubles when twice as much enzyme is used Prentice Hall c2002 Chapter 5 18 5.3 The Michaelis-Menten Equation • Maximum velocity (Vmax) is reached when an enzyme is saturated with substrate (high [S]) • At high [S] the reaction rate is independent of [S] (zero order with respect to S) • At low [S] reaction is first order with respect to S • The shape of a vo versus [S] curve is a rectangular hyperbola, indicating saturation of the enzyme active site as [S] increases Prentice Hall c2002 Chapter 5 19 Fig 5.3 Plots of initial velocity (vo) versus [S] (a) Each vo vs [S] point is from one kinetic run (b) Michaelis constant (Km) equals the concentration of substrate needed for 1/2 maximum velocity Prentice Hall c2002 Chapter 5 20 The Michaelis-Menten equation • Equation describes vo versus [S] plots • Km is the Michaelis constant Vmax[S] vo = Km + [S] Prentice Hall c2002 Chapter 5 21 A. Derivation of the Michaelis-Menten Equation • Derived from (1) Steady-state conditions: Rate of ES formation = Rate of ES decomposition (2) Michaelis constant: Km = (k-1 + k2) / k1 (3) Velocity of an enzyme-catalyzed reaction (depends upon rate of conversion of ES to E + P) vo = k2[ES] Prentice Hall c2002 Chapter 5 22 B. The Meanings of Km • Km = [S] when vo = 1/2 Vmax • Km @ k-1 / k1 = Ks (the enzyme-substrate dissociation constant) when kcat << either k1 or k-1 • The lower the value of Km, the tighter the substrate binding • Km can be a measure of the affinity of E for S Prentice Hall c2002 Chapter 5 23 Fig 5.4 Km and physiological substrate concentrations • Km values for enzymes are typically just above [S], so that the enzyme rate is sensitive to small changes in [S] Prentice Hall c2002 Chapter 5 24 5.4 Kinetic Constants Indicate Enzyme Activity and Specificity • Catalytic constant (kcat) - first order rate constant for conversion of ES complex to E + P • kcat most easily measured when the enzyme is saturated with S (region A on Figure 5) • Ratio kcat /Km is a second order rate constant for E+S E + P at low [S] concentrations (region B on Figure 5) Prentice Hall c2002 Chapter 5 25 Fig 5.5 Meanings of kcat and kcat/Km Prentice Hall c2002 Chapter 5 26 Table 5.1 Examples of catalytic constants kcat(s-1) Enzyme Prentice Hall c2002 Chapter 5 27 Values of kcat/Km • kcat/Km can approach rate of encounter of two uncharged molecules in solution (108 to 109M-1s-1) • kcat/Km is also a measure of enzyme specificity for different substrates (specificity constant) • rate acceleration = kcat/kn (kn = rate constant in the absence of enzyme) Prentice Hall c2002 Chapter 5 28 Prentice Hall c2002 Chapter 5 29 5.5 Measurement of Km and Vmax Fig 5.6 The doublereciprocal LineweaverBurk plot is a linear transformation of the Michaelis-Menten plot (1/vo versus 1/[S]) Prentice Hall c2002 Chapter 5 30 5.6 Kinetics of Multisubstrate Reactions Fig 5.7 Notations for bisubstrate reactions (a) Sequential (ordered or random) Ordered Random Prentice Hall c2002 Chapter 5 31 Fig. 5.7 (b) Bisubstrate reactions Prentice Hall c2002 Chapter 5 32 5.7 Reversible Enzyme Inhibition • Inhibitor (I) binds to an enzyme and prevents formation of ES complex or breakdown to E + P • Inhibition constant (Ki) is a dissociation constant EI E+I • There are three basic types of inhibition: Competitive, Uncompetitive and Noncompetitive • These can be distinguished experimentally by their effects on the enzyme kinetic patterns Prentice Hall c2002 Chapter 5 33 Fig 5.8 Reversible enzyme inhibitors (a) Competitive. S and I bind to same site on E (b) Nonclassical competitive. Binding of S at active site prevents binding of I at separate site. Binding of I at separate site prevents S binding at active site. Prentice Hall c2002 Chapter 5 34 Fig 5.8 (cont) (c) Uncompetitive. I binds only to ES (inactivates E) (d) Noncompetitive. I binds to either E or ES to inactivate the enzyme Prentice Hall c2002 Chapter 5 35 A. Competitive Inhibition • Inhibitor binds only to free enzyme (E) not (ES) • Substrate cannot bind when I is bound at active site (S and I “compete” for the enzyme active site) • Vmax is the same with or without I (high S can still saturate the enzyme even in the presence of I) • Apparent Km (Kmapp) measured in the presence of I is larger than KM (measured in absence of I) • Competitive inhibitors usually resemble the substrate Prentice Hall c2002 Chapter 5 36 Fig 5.9 Competitive inhibition. (a) Kinetic scheme. (b) Lineweaver-Burk plot Prentice Hall c2002 Chapter 5 37 Fig 5.10 Benzamidine competes with arginine for binding to trypsin Prentice Hall c2002 Chapter 5 38 B. Uncompetitive Inhibition • Uncompetitive inhibitors bind to ES not to free E • Vmax decreased by conversion of some E to ESI • Km (Kmapp) is also decreased • Lines on double-reciprocal plots are parallel • This type of inhibition usually only occurs in multisubstrate reactions Prentice Hall c2002 Chapter 5 39 Fig 5.11 Uncompetitive inhibition Prentice Hall c2002 Chapter 5 40 C. Noncompetitive Inhibition • Noncompetitive inhibitors bind to both E and ES • Inhibitors do not bind at the same site as S • Vmax decreases • Km does not change • Inhibition cannot be overcome by addition of S • Lines on double-reciprocal plot intersect on x axis Prentice Hall c2002 Chapter 5 41 Fig 5.12 Noncompetitive inhibition Prentice Hall c2002 Chapter 5 42 D. Uses of Enzyme Inhibition Fig 5.13 Comparison of a substrate (a) and a designed inhibitor (b) for the enzyme purine nucleoside phosphorylase Prentice Hall c2002 Chapter 5 43 5.8 Irreversible Enzyme Inhibition • Irreversible inhibitors form stable covalent bonds with the enzyme (e.g. alkylation or acylation of an active site side chain) • There are many naturally-occurring and synthetic irreversible inhibitors • These inhibitors can be used to identify the amino acid residues at enzyme active sites • Incubation of I with enzyme results in loss of activity Prentice Hall c2002 Chapter 5 44 Fig 5.14 Covalent complex with lysine residues • Reduction of a Schiff base forms a stable substituted enzyme Prentice Hall c2002 Chapter 5 45 Inhibition of serine protease with DFP • Diisopropyl fluorophosphate (DFP) is an organic phosphate that inactivates serine proteases • DFP reacts with the active site serine (Ser-195) of chymotrypsin to form DFP-chymotrypsin • Such organophosphorous inhibitors are used as insecticides or for enzyme research • These inhibitors are toxic because they inhibit acetylcholinesterase (a serine protease that hydrolyzes the neurotransmitter acetylcholine) Prentice Hall c2002 Chapter 5 46 Fig 5.15 • Reaction of DFP with Ser-195 of chymotrypsin Prentice Hall c2002 Chapter 5 47 Affinity labels for studying enzyme active sites • Affinity labels are active-site directed reagents • They are irreversible inhibitors • Affinity labels resemble substrates, but contain reactive groups to interact covalently with the enzyme Prentice Hall c2002 Chapter 5 48 Fig 5.16 Irreversible inhibition of triose phosphate isomerase • Bromo analog reacts with active site glutamate, and reduction with NaBH4 stabilizes the adduct Prentice Hall c2002 Chapter 5 49 5.9 Site-Directed Mutagenesis Modifies Enzymes • Site-directed mutagenesis (SDM) can be used to test the functions of individual amino acid side chains • One amino acid is replaced by another using molecular biology techniques • Bacterial cells can be used to synthesize the modified protein Prentice Hall c2002 Chapter 5 50 Practical applications of SDM • SDM is also used to change the properties of enzymes to make them more useful • Subtilisin protease was made more resistant to chemical oxidation by replacing Met-222 with Ala-222 (the modified subtilisin is used in detergents) • A bacterial protease was made more heat stable by replacing 8 of 319 amino acids Prentice Hall c2002 Chapter 5 51 5.10 Regulation of Enzyme Activity • Regulatory enzymes - activity can be reversibly modulated by effectors • Such enzymes are usually found at the first unique step in a metabolic pathway (the first “committed” step) • Regulation at this step conserves material and energy and prevents accumulation of intermediates Prentice Hall c2002 Chapter 5 52 Two Methods of regulation (1) Noncovalent allosteric regulation (2) Covalent modification • Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site • Allosteric inhibitors or activators bind to this site and regulate enzyme activity via conformational changes Prentice Hall c2002 Chapter 5 53 A. Phosphofructokinase-1 (PFK-1) Is an Allosteric Enzyme • PFK-1catalyzes an early step in glycolysis (an ATP-generating pathway for glucose degradation) • Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1 • ADP is an allosteric activator of PFK-1 Prentice Hall c2002 Chapter 5 54 Regulation of glycolysis by PFK-1 • When the ratio [PEP]/[ADP] is high, flux through glycolysis decreases at the PFK-1 step • When the ratio of [PEP]/[ADP] is low, PFK-1 is activated and glycolysis produces more ATP from ADP • Thus the concentrations of PEP and ADP act allosterically through PFK-1 to regulate the activity of the entire pathway Prentice Hall c2002 Chapter 5 55 Fig 5.17 Reaction catalyzed by PFK-1 Prentice Hall c2002 Chapter 5 56 Fig 5.18 Phosphoenolpyruvate (PEP) Prentice Hall c2002 Chapter 5 57 Effect of PEP and ADP on PFK-1 • Both PEP and ADP affect the binding of the substrate fructose 6-phosphate (F6P) to PFK-1 • PFK-1 exhibits a sigmoidal (S-shaped) vo versus [S] curve. This is due to the cooperativity of S binding. • Increasing ADP concentrations lowers the apparent Km for F6P without affecting Vmax (activates PFK) • The allosteric inhibitor PEP raises the apparent Km without changing Vmax Prentice Hall c2002 Chapter 5 58 Fig 5.19 Plots of initial velocity versus F6P for PFK-1 • ADP is an allosteric activator of PFK-1 and lowers the apparent Km without affecting Vmax • For a given F6P concentration the vo is larger in the presence of ADP Prentice Hall c2002 Chapter 5 59 Fig 5.20 Stereo views of PFK-1 from E. coli • PFK-1 is a tetramer of identical chains. Products (yellow and green), allosteric activator ADP (red) (a) Single chain ribbon diagram (b) Tetramer: 2 blue, 2 purple chains Prentice Hall c2002 Chapter 5 60 B. General Properties of Allosteric Enzymes 1. Activities of allosteric regulator enzymes are changed by inhibitors and activators (modulators) 2. Allosteric modulators bind noncovalently to the enzymes that they regulate 3. Regulatory enzymes possess quaternary structure (continued next slide) Prentice Hall c2002 Chapter 5 61 General properties of regulatory enzymes (cont) 4. Regulatory enzymes have at least one substrate which has a sigmoidal vo versus [S] curve (positive cooperativity of multiple substrate binding sites) 5. There is a rapid transition between the active (R) and inactive (T) conformations 6. Substrates and activators may bind only to the R state while inhibitors may bind only to the T state Prentice Hall c2002 Chapter 5 62 Rapid transition exists between R and T states • Addition of S increases concentration of the R state • Addition of I increases concentration of the T state • Activator molecules bind preferentially to R, leading to an increase in the R/T ratio Prentice Hall c2002 Chapter 5 63 Fig 5.21 Role of cooperativity of binding in regulation • Addition of modulators alters enzyme activity • Activators can lower Km, inhibitors can raise Km Prentice Hall c2002 Chapter 5 64 C. Two Theories of Allosteric Regulation Concerted theory - (symmetry-driven theory). • Only 2 conformations exist: R and T ( symmetry is retained in the shift between R and T states) • Subunits are either all R or all T • R has high affinity for S, T has a low affinity for S • Binding of S shifts the equilibrium toward all R state • Binding of I shifts the equilibrium toward all T state Prentice Hall c2002 Chapter 5 65 Sequential Theory (ligand-induced theory) • A ligand may induce a change in the structure of the subunit to which it binds • Conformational change of one subunit may affect the conformation of neighboring subunits • A mixture of both R (high S affinity) and T (low S affinity) subunits may exist (symmetry does not have to be conserved) Prentice Hall c2002 Chapter 5 66 Fig 5.22 Two models (a) Concerted model: subunits either all T state or all R state (b) Sequential model: Mixture of T subunits and R subunits is possible. Binding of S converts only that subunit from T to R Prentice Hall c2002 Chapter 5 67 Fig 5.23 Conformational changes during O2 binding to hemoglobin • Oxygen binding to Hb has aspects of both the sequential and concerted models Prentice Hall c2002 Chapter 5 68 D. Regulation by Covalent Modification • Interconvertible enzymes are controlled by covalent modification • Converter enzymes catalyze covalent modification • Converter enzymes are usually controlled themselves by allosteric modulators Prentice Hall c2002 Chapter 5 69 Fig 5.24 General scheme for covalent modification of interconvertible enzymes Prentice Hall c2002 Chapter 5 70 Fig 5.25 Pyruvate dehydrogenase regulation • Phosphorylation stabilizes the inactive state (red) • Dephosphorlyation stabilizes the active state (green) Prentice Hall c2002 Chapter 5 71 5.11 Multienzyme Complexes and Multifunctional Enzymes • Multienzyme complexes - different enzymes that catalyze sequential reactions in the same pathway are bound together • Multifunctional enzymes - different activities may be found on a single, multifunctional polypeptide chain Prentice Hall c2002 Chapter 5 72 Metabolite channeling • Metabolite channeling - “channeling” of reactants between active sites can occur when the product of one reaction is transferred directly to the next active site without entering the bulk solvent • Can greatly increase rate of a sequence of reactions • Can protect intermediates from solvent reactions • Channeling is possible in multienzyme complexes and multifunctional enzymes Prentice Hall c2002 Chapter 5 73