Enzymes

2009 W. H. Freeman and Company

Enzymes Key topics about enzyme function:

Physiological significance of enzymes Origin of catalytic power of enzymes Chemical mechanisms of catalysis Description of enzyme kinetics and inhibition

What are Enzymes?

Enzymes are catalytically active biological macromolecules
Most enzymes are globular proteins
some RNA (ribozymes, and ribosomal RNA) also catalyze reactions

Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s Study of enzymes has dominated biochemistry in the past and continues to do so

Classification of Enzymes
Each enzyme is assigned a four-part classification number and a systematic name, which identifies the reaction it catalyzes.

Binding of a substrate to an enzyme at the active site

The enzyme chymotrypsin, with bound substrate in red (PDB ID 7GCH). Some key active-site amino acid residues appear as a red splotch on the enzyme surface.

Transition State Theory

Slow reactions face significant activation barriers that must be surmounted during the reaction transition state theory is applicable for catalysis rate constants and free energies can be related G k T k = B exp h RT
The relationship between the rate constant k and activation energy G is inverse and exponential. A lower activation energy means a faster reaction rate.

Reaction coordinate diagram

Rate Acceleration
Enzymes affect reaction rate, not equilibria. A favorable equilibrium S P does not mean a detectable rate. The rate of a reaction depends on the activation energy G: a higher activation energy corresponds to a slower reaction. Catalysts enhance reaction rates by lowering activation energies.

Reaction coordinate diagram comparing enzyme-catalyzed and uncatalyzed reactions

How to Lower G?
Enzymes organize reactive groups into proximity
Uncatalyzed bimolecular reactions:
two free reactants single restricted transition state

Uncatalyzed unimolecular reactions:

flexible reactant rigid transition state conversion Catalyzed reactions: Enzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complex Entropy cost is paid during binding Rigid reactant complex transition state conversion

How to Lower G? Enzymes bind transition states best

The idea was proposed by Linus Pauling in 1946:
enzyme active sites are complimentary to the transition state of the reaction enzymes bind transition states better than substrates stronger interactions with the transition state as compared to the ground state lower the activation barrier

Largely H effect

Illustration of TS Stabilization Idea: Imaginary Stickase

How is TS Stabilization Achieved?

acid-base catalysis: give and take protons covalent catalysis: change reaction paths metal ion catalysis: use redox cofactors, pKa shifters electrostatic catalysis: preferential interactions with TS

Amino Acids in General Acid-Base catalysis

Covalent Catalysis: In Enzymes

Proteases and peptidases chymotrypsin, elastase, subtilisin reactive serine nucleophile Some aldehyde dehydrogenase glyceraldehyde-3phosphate dehydrogenase reactive thiolate nucleophile Aldolases and decarboxylases amine nucleophile Dehalogenases carboxylate nucleophile NH
2

HO N O

O S O O

O N O

What is Enzyme Kinetics?

Kinetics is the study of the rate at which compounds react Rate of enzymatic reaction is affected by Enzyme Substrate Effectors Temperature

Why Study Enzyme Kinetics?

Quantitative description of biocatalysis Determine the order of binding of substrates Elucidate acid-base catalysis Understand catalytic mechanism Find effective inhibitors Understand regulation of activity

Kinetics of Enzyme-Catalyzed Reactions

ES k-1 k-1 [ES] = k1 [E] [S] = E+S

KS is the dissociation constant of the enzyme-substrate complex.

k1

KS =

[E] [S] [ES]

k-1 k1

E+S

k1 k-1

ES

k2

E+P

Enzyme-Substrate Complex
[Etot] = [E] + [ES] f = k1 [E] [S] = k1 ([Etot] - [ES]) [S] d = k-1 [ES] + k2 [ES] f = d k1 ([Etot] - [ES]) [S] = (k-1 + k2) [ES]

([Etot] - [ES]) [S] [ES]

[ES] =

k-1 + k2 k1

= Km

[Etot] [S] Km + [S]

Michaelis-Menten Equation
The rate of formation of product is V0 = k2 [ES]

V0 =

k2 [Etot] [S] Km + [S]

When the enzyme is saturated, [ES]=[Etot], Vmax= k2[Etot], then

V0 =

Vmax [S] Km + [S]

Michaelis-Menten Equation
The final form in case of a single substrate is

E+S

k1 k-1

ES

k2

E+P

v=

k [ E ][ S ] K + [S ]
cat tot m

kcat (turnover number): how many substrate molecules can one enzyme molecule convert to product per second when the enzyme is saturated Km (Michaelis constant): an approximate measure of substrates affinity for enzyme Microscopic meaning of Km and kcat depends on the details of the mechanism

Initial velocities of enzyme-catalyzed reactions

Effect of Substrate Concentration

Ideal Rate:

V0 =
Deviations due to:

Vmax [S] Km + [S]

Limitation of measurements Substrate inhibition Substrate prep contains inhibitors Enzyme prep contains inhibitors

Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction

Dependence of initial velocity on substrate concentration

Determination of Kinetic Parameters

Nonlinear Michaelis-Menten plot should be used to calculate parameters Km and Vmax Linearized double-reciprocal plot is good for analysis of two-substrate data or inhibition
HOMEWORK: Worked example 6-1 pg. 199 Worked example 6-2 pg. 200; problem 8 pg. 230