Allosteric Enzymes • Allosteric enzymes have one or more allosteric sites • Allosteric sites are binding sites distinct from an enzyme’s active site or substrate-binding site • Molecules that bind to allosteric sites are called effectors or modulators • Binding to allosteric sites alters the activity of the enzyme. This is called cooperative binding. Allosteric enzymes display sigmoidal plot of Vo vs [S] • Effectors may be positive or negative • Effectors may be homotropic or heterotropic • Regulatory enzymes of metabolic pathways are allosteric enzymes (eg: feedback inhibition)

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

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

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.

Enzyme Inhibition

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

Competitive Inhibition

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

V max unaltered, Km increased

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)

Mixed (Noncompetitive) Inhibition

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

Km unaltered, V max decreased

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).

Uncompetitive Inhibition

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

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

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

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

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

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

Factors Affecting Enzyme Kinetics

Effects of pH - enzymes have ionic groups on their active sites. - 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.

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

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

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

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