1 / 28

Chapter 14 Rates of Enzymatic Reactions

Chapter 14 Rates of Enzymatic Reactions. Reading: V&V pp. 472-487. Chymotrypsin with bound substrate. Enzyme Kinetics. Enzymes accelerate reactions by lowering the free energy of activation Enzymes do this by binding the transition state of the reaction better than the substrate.

humphrey
Download Presentation

Chapter 14 Rates of Enzymatic Reactions

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Chapter 14Rates of Enzymatic Reactions Reading: V&V pp. 472-487 Chymotrypsin with bound substrate

  2. Enzyme Kinetics • Enzymes accelerate reactions by lowering the free energy of activation • Enzymes do this by binding the transition state of the reaction better than the substrate Several terms to know: • rate or velocity • rate constant • rate law • order of a reaction • molecularity of a reaction

  3. The Michaelis-Menten Equation • Louis Michaelis and Maude Menten's theory • It assumes the formation of an enzyme-substrate complex • It assumes that the ES complex is in rapid equilibrium with free enzyme • Breakdown of ES to form products is assumed to be slower than (1) formation of ES and (2) breakdown of ES to re-form E and S

  4. The dual nature of the Michaelis-Menten equation Combination of zero-order and 1st-order kinetics

  5. k1 k-1 E + SESE + P k2 (k-2 is insignificant early in rxn) Vo = k2[ES] Rate of ES formation = k1[E][S] = k1([Etotal] - [ES]) [S] Rate of ES breakdown = k-1[ES] + k2[ES] k1([Etotal] - [ES]) [S] = k-1[ES] + k2[ES] (steady state assumption)

  6. k1[Etotal][S] - k1[ES][S] = ( k-1+ k2)[ES] k1[Etotal][S] = (k1[S] + k-1+ k2)[ES] [ES] = [Etotal][S] ____________ KM+ [S] [Etotal][S] ________________________ [S] + (k2 + k-1) ___________ k1 = k2 [Etotal][S] ____________ KM+ [S] Vo = k2[ES] Vo = Vo = Vmax when [Etotal] = [ES] (at saturation) Therefore Vmax = k2[Etotal] Vmax[S] ____________ KM+ [S] Vo =

  7. Vmax[S] _________ Km + [S] Vo = The dual nature of the Michaelis-Menten equation Combination of zero-order and first-order kinetics • When [S] is low, the equation for rate is first order in [S] • When [S] is high, the equation for rate is zero-order in [S] • The Michaelis-Menten equation describes a rectangular hyperbolic dependence of Vo on [S]

  8. Enzyme Kinetics:Michaelis-Menton Equation Vmax[S] Vo = ____________ KM + [S] KM = [S] when Vo = Vmax _____ 2 From Lehninger Principles of Biochemistry

  9. The following data were obtained in a study of an enzyme known to follow Michaelis Menten kinetics: V0 Substrate added (mmol/min) (mmol/L) ————————————— 216 0.9 323 2 435 4 489 6 647 2,000 ————————————— Calculate the Km for this enzyme. Km is the substrate concentration that corresponds to Vmax 2 Without graphing Vmax = 647 Vmax /2 = 647 / 2 = 323.5 Km = 2 mmol/L

  10. Understanding Km • Km is a constant • Km is a constant derived from rate constants • Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S • Small Km means tight binding; high Km means weak binding Enzyme Substrate Km (mM) Glutamate dehydrogenase NH4+ 57 Glutamate 0.12 Carbonic anhydrase CO2 12

  11. Understanding Vmax The theoretical maximal velocity • Vmax is a constant • Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality • To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate • Vmax is asymptotically approached as substrate is increased

  12. The turnover number (also known as the molecular activity of the enzyme) A measure of its maximal catalytic activity • kcat, the turnover number, is the number of substrate molecules converted to product per enzyme molecule per unit of time, when E is saturated with substrate. • If the M-M model fits, k2 = kcat kcat = Vmax/Et • Values of kcat range from less than 1/sec to many millions per sec Turnover number comparison Catalase 40,000,000 sec-1 Lysozyme 0.5 sec-1

  13. Catalytic efficiency of an enzyme Name for kcat/Km • An estimate of "how perfect" the enzyme is • kcat/Km is an apparent second-order rate constant • It measures how the enzyme performs when S is low • Catalytic efficiency cannot exceed the diffusion limit - the rate at which E and S diffuse together • WT and a mutant protein kcat/Km comparision WT sulfite oxidase 1.1 Mutant R160K 0.015

  14. Use linear plot and intercepts to determine Km and Vmax 1 KM 1 ______ = _______ +______ Vo Vmax[S] Vmax Double-Reciprocal orLineweaver-Burk Plot From Lehninger Principles of Biochemistry

  15. pH must be specified!

  16. Enzyme Inhibitors Reversible versus Irreversible • Reversible inhibitors interact with an enzyme via noncovalent associations • Irreversible inhibitors interact with an enzyme via covalent associations

  17. Classes of Inhibition Two real, one hypothetical • Competitive inhibition - inhibitor (I) binds only to E, not to ES • Uncompetitive inhibition - inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition • Noncompetitive (mixed) inhibition - inhibitor (I) binds to E and to ES

  18. Inhibitor (I) binds only to E, not to ES Inhibitor (I) binds only to ES, not to E. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition Enzyme Inhibition Inhibitor (I) binds to E and to ES. From Lehninger Principles of Biochemistry

  19. CompetitiveUncompetitiveNoncompetitive InhibitionInhibition(Mixed) Inhibition Kmchanges while Vmax does not Km and Vmax both change Km and Vmax both change From Lehninger Principles of Biochemistry

  20. Succinate dehydrogenase is a classic example of competitive inhibition Malonate is a strong competitive inhibitor of succinate dehydrogenase From Lehninger Principles of Biochemistry

  21. Competitive Inhibition Kmchanges while Vmax does not 1/[S] + I 1/V No I -1 / Km -1 / Kmapp Where Kmapp = a Km a = 1 + [I] KI

  22. Uncompetitive inhibition 1/[S] + I a’/Vmax 1/V No I -a’ / Km 1/Vmax -1 / Km a‘ = 1 + [I] KI

  23. Effects of Inhibitors on the parameters of Michaelis-Menten Equation Type of inhibition Vmaxapp KMapp No inhibitor Vmax KM Competitive VmaxaKM Uncompetitive Vmax/a’ KM/a’ Noncompetitive (Mixed) Vmax/a’ aKM/a’ a = 1 + [I] a’ = 1 + [I] KI KI’

  24. Regulation of enzymatic activity • Two ways that this may occur: • Control of enzyme availability • Depends on rate of enzyme synthesis & degradation • Control of enzyme activity • Enzyme-substrate binding affinity may vary with binding of small molecules called allosteric effectors (ex: BPG for Hb) • Allosteric mechanisms can cause large changes in enzymatic activity

  25. 1. Allosteric enzymes 2. Regulation by covalent modification Regulatory Enzymes important in controlling flux through metabolic pathways From Lehninger Principles of Biochemistry

  26. Regulation by Feedback Inhibition Conversion of L-threonine to L-isoleucine catalyzed by a sequence five enzymes, E1-E5 L-isoleucine is an inhibitory allosteric modulator of E1 From Lehninger Principles of Biochemistry

More Related