Enzyme Kinetics

Kinetics of Enzyme-Catalyzed Reactions: Michaelis-Menten Kinetics Enzyme Inhibition Kinetics of Two-Substrate Reactions Ribozymes and Abzymes

How Enzymes Work

Enzyme-catalyzed reactions are characterized by the formation of a complex between the enzyme and its substrate (the ES complex) Substrate binding occurs in a pocket on the enzyme called the active site Enzymes accelerate reactions by lowering the free energy of activation DG. The equilibrium of the reaction remains unaffected by the enzyme Enzymes do this by binding the transition state of the reaction better than the substrate

Where Does the Energy Come From?

Enzyme-substrate interactions are predominantly non-covalent ionic H-bonds hydrophobic The active site is structured so that more of these interactions occur in the transition state These bonds account for a significant part of the energy required to reduce DG The rest comes from the folding free energy of the enzyme in its ES conformation

The Michaelis-Menten Equation

can we find the relationship between rate and [S]? Step 1. Formation of the ES complex: forward rate v1 = k1[S][E] backward rate v-1 = k-1[ES] k1 S+E ES k-1 This is an equilibrium where the equilibrium constant K = k1/k-1 Step 2. Formation of products: forward rate v2 = k2[ES] backward rate v-2 = k-2[E][P] ES k2 k-2 Breakdown of ES to form products is assumed to be slower than formation of ES (v1) or breakdown of ES to re-form E and S (v-1) E+P

Assumption 1
Only consider early times in the reaction [P] is very small v-2 is negligible step 2 is essentially irreversible k1 k2 E+S ES k-1

E+P

So the initial rate v0 = k2[ES] We can easily measure initial rates, but [ES] is very difficult to measure

An Expression for [ES]

Recall that [E]total = [E] + [ES] How fast is the enzyme-substrate complex formed and broken down? k1 k2 E+S ES E+P k-1 Step 1: rate of formation of ES = k1[S]([E]total - [ES]) Steps 1 and 2: rate of breakdown of ES = k-1[ES] + k2[ES]

Assumption 2
Briggs-Haldane Steady State Assumption rate of ES formation = rate of ES breakdown k1[S]([E]total - [ES]) = k-1[ES] + k2[ES] [ES] = k1[S][E]total k-1 + k2 + k1[S] [S][E]total [S] + k-1 + k2 k1

[ES] =

Substitute for [ES] in the initial rate equation v0 = k2[ES] [ES] = [S][E]total [S] + k-1 + k2 k1

v0 =

k2[S][E]total [S] + k-1 + k2 k1

Assumption 3
[S] >> [E]total The enzyme is saturated with substrate [ES] = [E]total So we can define a maximum rate Vmax where Vmax = k2[E]total v0 = Vmax[S] [S] + k-1 + k2 k1

Now group the constants: Km = (k-1 + k2)/k1

v0 = Vmax[S] Km + [S]
The Michaelis-Menten equation is the rate equation for a one-substrate enzymecatalyzed reaction It quantitatively relates the initial rate, the maximum rate, and the initial substrate concentration to the Michaelis constant Km

Understanding Km
The "kinetic activator constant" Km is a constant with units M 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

Understanding Vmax
theoretical maximal velocity Vmax is a constant with units s-1 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 [S] is increased

The Dual Nature of the Michaelis-Menten Equation

combination of zero and 1st-order kinetics When [S] is low, the equation for rate is 1st 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 v0 on [S]

The Turnover Number

a measure of 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 = Vmax/Etotal Values of kcat range from less than 1/sec to many millions per sec

Catalytic Efficiency
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 The upper limit for kcat/Km is the diffusion limit - the rate at which E and S diffuse together (108 to 109 s-1)

Linear Plots of the Michaelis-Menten Equation

be able to derive these relationships Lineweaver-Burk (double reciprocal) Hanes-Woolf Hanes-Woolf is best Smaller and more consistent errors across the plot

Lineweaver-Burk Plot

Hanes-Woolf Plot

Lineweaver-Burk Plots
Vmax 800 v0 (s-1) 600 400 200 Vmax = 1000 s-1 Km= 1x10-4 M

1/v0 (M -1s) 2x10 -3 1.5x10 -3 1x10 -3 5x10 -4 slope = Km/Vmax

1/V max

2x10 -4

4x10 -4

6x10 -4

8x10 -4

10 -3

-1/Km

-5000

0 1/[S] (M -1)

5000

10000

[S] (M)

Note how the straight line fit can be weighted by unreliable values at small [S] and v0 The Lineweaver-Burk plot is useful for comparing inhibition mechanisms

Enzyme Inhibitors
reversible versus irreversible Irreversible inhibitors interact with an enzyme via covalent associations Nerve agents like sarin are irreversible inhibitors of acetylcholine esterase Reversible inhibitors interact with an enzyme via noncovalent associations For therapeutic drug design were almost always interested in reversible inhibitors

Reversible Inhibition
Competitive inhibition - I binds to E, not to ES Substrate and inhibitor compete for the active site Vmax unchanged In principle we can always add enough substrate to produce saturated ES (though not in practice) Km becomes Km + Km[I]/KI where KI is the equilibrium constant for E + I = EI The apparent ES association gets weaker (Km gets larger)

Reversible Inhibition
Noncompetitive inhibition - I binds either to ES or to E and ES Pure noncompetetive inhibition (uncommon) The binding of I to E has no effect on the binding of S E + I = EI and ES + I = ESI have the same KI S and I bind at different sites and the active site is unaffected by I Km is unchanged The inhibitor is not binding at the active site, so Vmax cannot be recovered by raising [S] Vmax becomes Vmax/(1 + [I]/KI) I only affects k2, for example by slowing product release

Reversible Inhibition

Noncompetitive inhibition - I binds either to ES or to E and ES Mixed noncompetetive inhibition (common) Binding of I by E influence binding of S E + I = EI has KI ES + I = ESI has KI KI ! KI Both Km and Vmax are changed Km becomes (Km + Km[I]/KI)/(1 + [I]/KI) Vmax becomes Vmax/(1 + [I]/KI) Inhibitor binds close to the active site, or by binding elsewhere on E has an influence on the active site

Reversible Inhibition

Uncompetitive inhibition - I binds only to ES Few examples known, eg Roundup is an uncompetetive inhibitor of 3-enolpyruvlyshikimate-5-P synthase ES + I = ESI has KI Both Km and Vmax are changed Km becomes Km/(1 + [I]/KI) Vmax becomes Vmax/(1 + [I]/KI)

1 + [I]/KI Vmax

-1 + [I]/KI Km

-1 Km

Irreversible Inhibition

Suicide substrates Substrate analogs that become highly reactive through normal action of E Form covalent bonds at or near the active site causing irreversible inhibition Eg penicillin reacts with an active-site serine

Glycoprotein peptidase crosslinks cell wall peptidoglycans in gram-negative bacteria Weakened cell walls halt bacterial growth

Enzyme Inhibition: Summary

Michaelis-Menten kinetics defined by Vmax and Km Remember how Vmax and Km change with different classes of inhibitors (Table 13.6 on p422) Substitute the new Km and Vmax into the Michaelis-Menten equation to get the relationship with v0 Substitute for -1/Km and 1/Vmax to use double reciprocal plots to calculate kinetic parameters (eg KI)

Bimolecular Reactions
The general case is A + B P+Q Possible routes 1. E + A + B EAB EPQ E+P+Q sequential, or single displacement mechanism may be random or ordered 2a. E + A EA EP E + P b. E + B EB EQ E+Q ping-pong, or double displacement mechanism

Single Displacement Kinetics

Single Displacement Kinetics

Random Single Displacement Reactions The conversion of EAB to EPQ is rate-determining

A+ E

EA

EP

P+ E

For the special case in which A has no influence on the binding of B (and vice versa) the lines on a double reciprocal plot intersect on the 1/[A] axis Km doesnt change with [A] or [B]

EAB

EPQ

B + E

EB

EQ

Q+ E

Single Displacement Kinetics

Ordered Single Displacement Reactions A binds to E, then B binds to EA. A is the leading substrate B does not bind E

E A A E

EA B

EP Q

E P

Double Displacement Kinetics

Proceed via formation of a covalent enzyme intermediate

E Q

E B

Ribozymes and Catalytic Antibodies Ribozymes - segments of RNA that display enzyme activity in the absence of protein Examples: RNase P and peptidyl transferase Catalytic antibodies - antibodies raised to bind the transition state of a reaction of interest

Catalytic Antibodies