1 Chapter 3 - Enzymes

2 Enzyme Inhibition Enzyme can be inhibited by inhibitors. Inhibitors are tools to scientists to understand enzymes. Inhibitors are also in many cases pharmaceutical reagents against diseases; Inhibitors inhibit enzyme function by binding with enzymes. The binding reaction can be either reversible or irreversible; Irreversible inhibitors combine irreversibly with enzymes. Irreversible inhibitors associate with enzymes through covalent interactions. Thus the consequences of irreversible inhibitors is to decrease in the concentration of active enzymes.

3 Enzyme Inhibition (cont.) Several drugs in current medical use are irreversible inhibitors of enzymes, or mechanism-based enzyme inactivators. For instance, penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to cross-link the peptidyglycan chains during synthesis of bacterial cell walls. Reversible inhibitors associate with enzymes through non- covalent interactions. Reversible inhibitors include three kinds: –Competitive inhibitors; –Non-competitive inhibitors; –Un-competitive inhibitors (all of them under rapid equilibrium assumption)

Useful animations on enzyme inhibition kinetics – 8/student/animations/enzyme_inhibition/index. htmlhttp:// 8/student/animations/enzyme_inhibition/index. html – 90/animations/enzyme_inhibition/enzyme_inhi bition.htmhttp:// 90/animations/enzyme_inhibition/enzyme_inhi bition.htm 4

5 Competitive Inhibition In competitive inhibition, the inhibitor binds to the substrate binding site as shown (part b), thus preventing substrate binding. Competitive inhibition causes the Km value to increase, but does not affect Vmax. A competitive inhibitor binds reversibly to the enzyme, preventing the binding of substrate. On the other hand, binding of substrate prevents binding of the inhibitor, thus substrate and inhibitor compete for the enzyme.

6 Competitive Inhibition k 3 k -3 K I

7 Competitive Inhibition v [S][S] v max KmKm 1/[S] 1/v 1/v max -1/K’ m Slope=K’ m /v max k1k1 K -1 k2k2 k3k3 k -3 inhibitor K’ m (1+[I]/K I ) -1/(K’ m (1+[I]/K I )) no inhibitor Slope= K’ m (1+[I]/K I )/v max [I] increases

8 Non-competitive inhibition Non-competitive inhibitors never bind to the active site, but to other parts of the enzyme that can be far away from the substrate binding site, consequently, there is no competition between the substrate and inhibitor for the enzyme. The extent of inhibition depends entirely on the inhibitor concentration and will not be affected by the substrate concentration.

9 Noncompetitive Inhibition

10 Noncompetitive Inhibition k1k1 k -1 k2k2 KIKI K’ m unchanged V m,app decreases KIKI inhibitor 1/[S] 1/v 1/v max -1/K’ m Slope=K m /v max Slope= K’ m (1+[I]/K I )/v max v [S][S] v max K’ m V max /(1+[I]/K I ) k1k1 k -1 No inhibitor [I] increases (1+[I]/K I )/V max

11 Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme- substrate complex, not to the free enzyme, the EIS complex is catalytically inactive. This mode of inhibition is rare. Uncompetitive inhibition causes a decrease in V max,app and K’ m,app value. Uncompetitive Inhibition

12 k1k1 k -1 k2k2 K’ m decreases v max decreases Slope unchanged inhibitor KIKI 1/[S] 1/v 1/v max -1/K m Slope=K’ m /v max (1+ K I /[I])/V max Slope= K’ m /v max v [S][S] v max K’ m K’ m /(1+ K I /[I]) V max /(1+K I /[I]) Uncompetitive Inhibition How to derive this equation?? - (1+ K I /[I])/K’ m [I] increases

Enzyme Inhibition (Mechanism) CompetitiveNon-competitive Uncompetitive E E Different site Compete for active site Inhibitor Substrate Cartoon Guide Equation and Description I [ I ] binds to free [E] only, and competes with [S]; increasing [S] overcomes I Inhibition by [ I ]. I [ I ] binds to free [E] or [ES] complex; Increasing [S] can I not overcome [ I ] inhibition. I [ I ] binds to [ES] complex only, increasing [S] favors I the inhibition by [ I ]. E + S → ES → E + P + I ↓ I E I ← ↑ E + S → ES → E + P + + II I I ↓ II E I + S →E I S ← ↑ ↑ E + S → ES → E + P + I ↓ I E I S ← ↑ X Juang RH (2004) BCbasics

KmKm Enzyme Inhibition (Plots) CompetitiveNon-competitive Uncompetitive Direct Plots Double Reciprocal V max KmKm Km’Km’[S], mM vovo vovo II KmKm V max I Km’Km’ V max ’ V max unchanged K m increased V max decreased K m unchanged Both V max & K m decreased I 1/[S]1/K m 1/v o 1/ V max I Two parallel lines I Intersect at X axis 1/v o 1/ V max 1/[S]1/K m 1/[S]1/K m 1/ V max 1/v o Intersect at Y axis = Km’= Km’ Juang RH (2004) BCbasics

15 High substrate concentration may cause inhibition in some enzymatic reactions. Substrate inhibition and uncompetitive inhibition have similar mechanisms by inhibiting on ES complex. Substrate Inhibition

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17 k1k1 k -1 k2k2 Substrate Inhibition

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19 Substrate Inhibition At low substrate concentration, No inhibition effect is observed. At high substrate concentration, Inhibition is dominant.

20 Substrate Inhibition The substrate concentration resulting in the maximum reaction rate can be determined by setting dv/d[S]=0. The [S] max is given by * a 2 + b 2 ≥ 2ab and a 2 + b 2 = 2ab when a = b

21 Summary of Classes of Inhibitors Competitive inhibition - inhibitor (I) binds only to Enzyme, not to ES complex. Noncompetitive inhibition - inhibitor (I) binds either to Enzyme and/or to ES complex. Uncompetitive inhibition - inhibitor (I) binds only to ES complex, not to Enzyme. This is a hypothetical case that has never been documented for a real enzyme, but which makes a useful contrast to competitive inhibition. Substrate inhibition - Substrate inhibition and uncompetitive inhibition have similar mechanisms by inhibiting on ES complex.

22 Summary of Inhibition Kinetics

23 Review 3 Batch Kinetics

24 Review 3 Enzyme Inhibition. Reversible inhibitors include three kinds: –Competitive inhibitors; –Non-competitive inhibitors; –Un-competitive inhibitors Competitive Inhibition the inhibitor binds to the substrate binding site as shown, thus preventing substrate binding. (Km value to increase, but does not affect Vmax.)

25 Review 3 Non-competitive inhibition Non-competitive inhibitors never bind to the active site, but to other parts of the enzyme that can be far away from the substrate binding site

26 Review 3 Uncompetitive inhibition Uncompetitive inhibition occurs when the inhibitor binds only to the enzyme-substrate complex, not to the free enzyme, the EIS complex is catalytically inactive.

27 Review 3 Substrate inhibition Substrate inhibition and uncompetitive inhibition have similar mechanisms by inhibiting on ES complex.. The substrate concentration resulting in the maximum reaction rate can be determined by setting dv/d[S]=0.

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29 pH Effects on Enzyme Kinetics Certain enzymes have ionic groups on their active site. These ionic groups must be in a suitable form to function. pH change may alter the three-dimensional shape of enzyme, then affect enzyme activity (K m, V m and stability). Enzyme is only active over a certain pH range. In some cases, the substrate may contain ionic groups, and the pH of medium affects the affinity of the substrate to the enzyme.

30 pH Effects on Enzyme Kinetics

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32 Optimum pH *

33 Determination of optimum pH of enzyme The pH-activity profiles of two enzymes. (A) Trypsin; (B) Cholinesterase.

34 Temperature Effects on Enzyme Kinetics

35 Temperature Effects on Enzyme Kinetics “Enzyme denaturation by temperature is much faster than enzyme activation. e.g. a rise in temperature from 30ºC to 40ºC results in a 1.8-fold increase in enzyme activity but a 41-fold increase in enzyme denaturation” Denaturation constant The activation energies are within the 4 to 20 kcal/g mol range (mostly 11 kcal/g mol) Mostly about 70 kcal/g mol

36 Non-protein enzyme: Ribozymes It was assumed that all enzymes are proteins until 1982 when Thomas Cech and Sydney Altman discovered catalytic RNAs (Nobel, 1989 in Chemistry) Catalytic RNA, or ribozymes, satisfy several enzymatic criteria: substrate specificity, enhance reaction rate, and not change free energy from reaction Several known ribozymes: –RNase P: catalyzes cleavage of precursor tRNA molecules into mature tRNAs; –Group I, II introns: catalyze their own splicing (cleaving and ligating); –Ribosome: catalyzes peptidyl transfer reaction

37 pH Effects on Enzyme Kinetics Review 4

38 Temperature Effects on Enzyme Kinetics Review 4

39 Enzyme Reactor with simple kinetics Bioreactor-a device within which biochemical transformation are caused by the action of enzymes or living cells. Bioreactor is frequently called a fermenter, but we call bioreactor employing enzymes an enzyme reactor to distinguish it from the bioreactor which employs living cells, the fermenter.

40 Batch Reactor No flow-in and flow-out Equipped with an agitator to mix the reactants The pH is maintained by employing either a buffer solution or a pH controller. An ideal batch reactor is assumed to be well mixed so that the contents are uniform in composition at all times V, [S] [S]=[S] 0 at t=0

41 Batch Reactor Substrate Mass Balance: Accumulation=flow in –flow out + generation V, [S] [S]=[S] 0 at t=0 Like enzyme batch kinetics

42 Batch Reactor V, [S] [S]=[S] 0 at t=0

43 Continuous Stirred-Tank Reactor (CSTR) CSTR is an ideal reactor which is based on the assumption that the reactor contents are well mixed. The concentrations of various components of the outlet stream are assumed to be the same as the concentration of these components in the reactor. Continuous operation can increase the productivity. Easy to automate in order to reduce labor costs. V, [S] F- the flow rate [S] 0 F [S]

44 Continuous Stirred-Tank Reactor (CSTR) Substrate Mass Balance: Accumulation=flow in –flow out + generation V, [S] At steady state, d[S]/dt = 0 F [S] 0 F [S]