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Allosteric enzymes

Allosteric enzymes. Allosteric enzymes tend to be multi-sub unit proteins The reversible binding of an allosteric modulator (here a positive modulator M) affects the substrate binding site. Kinetics. Models. Cooperation. v o. (+). [S]. (+). v o. (+). [S]. v o. (-). (-). [S].

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Allosteric enzymes

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  1. Allosteric enzymes • Allosteric enzymes tend to be multi-sub unit proteins • The reversible binding of an allosteric modulator (here a positive modulator M) affects the substrate binding site

  2. Kinetics Models Cooperation vo (+) [S] (+) vo (+) [S] vo (-) (-) [S] Mechanism and Example of Allosteric Effect Allosteric site R = Relax (active) Homotropic (+) Concerted Allosteric site A Heterotropic (+) Sequential X Heterotropic (-) Concerted T = Tense (inactive) I X X

  3. Enzyme Inhibitors • Specific enzyme inhibitors regulate enzyme activity and help us understand mechanism of enzyme action. (Denaturing agents are not inhibitors) • Irreversible inhibitors form covalent or very tight permanent bonds with aa at the active site of the enzyme and render it inactive. 3 classes: groupspecific reagents, substrate analogs, suicide inhibitors • Reversible inhibitors form an EI complex that can be dissociated back to enzyme and free inhibitor. 3 groups based on their mechanism of action: competitive, non-competitive and uncompetitive.

  4. Enzyme Inhibition

  5. Competitive inhibitors • Compete with substrate for binding to enzyme • E + S = ES or E + I = EI . Both S and I cannot bind enzyme at the same time • In presence of I, the equilibrium of E + S = ES is shifted to the left causing dissociation of ES. • This can be reversed / corrected by increasing [S] • Vmax is not changed, KM is increased by (1 + I/Ki) • Eg: AZT, antibacterial sulfonamides, the anticancer agent methotrexate etc

  6. Competitive Inhibition

  7. Kinetics of competitive inhibitor Increase [S] to overcome inhibition Vmax attainable, Km is increased Ki = dissociation constant for inhibitor

  8. Vmax unaltered, Km increased

  9. Non-competitive Inhibitors • Inhibitor binding site is distinct from substrate binding site. Can bind to free enzyme E and to ES • E + I = EI, ES + I = ESI or EI + S = ESI • Both EI and ESI are enzymatically inactive • The effective functional [E] (and [S]) is reduced • Reaction of unaffected ES proceeds normally • Inhibition cannot be reversed by increasing [S] • KM is not changed, Vmax is decreased by (1 + I/Ki)

  10. Mixed (Noncompetitive) Inhibition

  11. Kinetics of non-competitive inhibitor Increasing [S] cannot overcome inhibition Less E available, Vmax is lower, Km remains the same for available E

  12. Km unaltered, Vmax decreased

  13. Uncompetitive Inhibitors • The inhibitor cannot bind to the enzyme directly, but can only bind to the enzyme-substrate complex. • ES + I = ESI • Both Vmax and KM are decreased by (1+I/Ki).

  14. Uncompetitive Inhibition

  15. Km’ E + S ES E + P k2 + S ES2 KS1 Substrate Inhibition • Caused by high substrate concentrations

  16. Substrate Inhibition • At low substrate concentrations [S]2/Ks1<<1 and inhibition is not observed • Plot of 1/v vs. 1/[S] gives a line • Slope = K’m/Vm • Intercept = 1/Vm

  17. Substrate Inhibition • At high substrate concentrations, K’m/[S]<<1, and inhibition is dominant • Plot of 1/v vs. [S] gives a straight line • Slope = 1/KS1· Vm • Intercept = 1/Vm

  18. 1/V I>0 I=0 1/Vm -1/Km -1/Km,app 1/[S] 1/V 1/V 1/V I>0 I>0 I=0 I=0 1/Vm,app 1/Vm,app 1/Vm 1/Vm 1/Vm -1/Km 1/[S] -1/Km 1/[S] -1/Km,app -1/Km 1/[S] Competitive Uncompetitive Substrate Inhibition Non-Competitive

  19. E + S→ES→E + P + I ↓ EI E + S→ES→E + P + + II ↓ ↓ EI+S→EIS E + S→ES→E + P + I ↓ EIS ← ← ← ↑ ↑ ↑ ↑ Enzyme Inhibition (Mechanism) Uncompetitive Non-competitive Competitive E Substrate E X Cartoon Guide Compete for active site Inhibitor Different site Equation and Description [I] binds to free [E] only, and competes with [S]; increasing [S] overcomes Inhibition by [I]. [I] binds to [ES] complex only, increasing [S] favors the inhibition by [I]. [I] binds to free [E] or [ES] complex; Increasing [S] can not overcome [I] inhibition.

  20. Uncompetitive Competitive Non-competitive Vmax Vmax vo Vmax’ Vmax’ I Direct Plots Km [S], mM Km’ Km [S], mM 1/vo 1/vo 1/vo I I Double Reciprocal Two parallel lines Intersect at X axis Intersect at Y axis 1/Vmax 1/Vmax 1/Vmax 1/Km 1/[S] 1/Km 1/[S] 1/Km 1/[S] Enzyme Inhibition (Plots) Vmax vo I I Km Km’ [S], mM =Km’ Vmax unchanged Km increased Vmax decreased Km unchanged Both Vmax & Km decreased I

  21. Factors Affecting Enzyme Kinetics

  22. Effects of pH - on enzymes - enzymes have ionic groups on their active sites. - Variation of pH changes the ionic form of the active sites. - pH changes the three-Dimensional structure of enzymes. - on substrate - some substrates contain ionic groups - pH affects the ionic form of substrate affects the affinity of the substrate to the enzyme.

  23. Effects of Temperature • Reaction rate increases with temperature up to a limit • Above a certain temperature, activity decreases with temperature due to denaturation • Denaturation is much faster than activation • Rate varies according to the Arrhenius equation Where Ea is the activation energy (kcal/mol) [E] is active enzyme concentration

  24. Factors Affecting Enzyme Kinetics • Temperature - on the rate of enzyme catalyzed reaction k2=A*exp(-Ea/R*T) T k2 - enzyme denaturation T Denaturation rate: kd=Ad*exp(-Ea/R*T) kd: enzyme denaturation rate constant; Ea: deactivation energy

  25. REFERENCES • Michael L. Shuler and Fikret Kargı, Bioprocess Engineering: Basic Concepts (2 nd Edition),PrenticeHall, New York, 2002. • 1. James E. Bailey and David F. Ollis, Biochemical Engineering Fundementals (2 nd Edition), McGraw-Hill, New York, 1986.

  26. www-nmr.cabm.rutgers.edu/academics/biochem694/2005BioChem412/Biochem.412_2005_Lect.18.ppt – • juang.bst.ntu.edu.tw/BCbasics/Animation.htm - 37k – • www.saburchill.com/IBbiology/chapters03/images/ENZYME%20INHIBITION.ppt – • http://www.wiley.com/college/pratt/0471393878/student/animations/enzyme_inhibition/index.html

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