Enzyme Mechanisms

Types of Enzymes

1. Oxidoreductases catalyze oxidation-reduction reactions.2. Transferases catalyze transfer of functional groups from

one molecule to another.3. Hydrolases catalyze hydrolytic cleavage.4. Lyases catalyze removal of a group from or addition of a

group to a double bond, or other cleavages involving electron rearrangement.

5. Isomerases catalyze intramolecular rearrangement.6. Ligases catalyze reactions in which two molecules are

joined.

Two Models for Enzyme-Substrate Interaction

Induced Conformational Change in Hexokinase

Coenzymes

Example of a Coenzyme Involved in Oxidation/Reduction Reactions

(nicotinamide adenine dinucleotide)

Hydride (H:-) transfer

Stereospecificity of Yeast Alcohol Dehydrogenase

pro-R

pro-S

ethanol acetaldehyde

Vennesland and Westheimer, 1950

pro-S

pro-R

Stereospecificity Conferred by an Enzyme

Catalytic Mechanisms

1. Acid-base catalysis2. Covalent catalysis3. Metal ion catalysis4. Electrostatic catalysis5. Proximity and orientation effects6. Preferential binding to transition state

(transition state stabilization)

Acid-Base Catalysis

Keto-Enol Tautomerism:Uncatalyzed vs. Acid- or Base-

Catalyzed

Example of Acid-Base Catalysis: Bovine Pancreatic RNase A

Covalent Catalysis: Nucleophiles and Electrophiles

Protonated

Example of Covalent Catalysis:Decarboxylation of Acetoacetate

Lysine side chain -amino group on enzyme is nucleophile in attack on substrate.

Electrophilic “electron sink”

Example of Metal Ion Catalysis: Carboxypeptidase A

Example of Metal Ion Catalysis: Carbonic Anhydrase

Carbonic anhydrase catalyzes the reaction:CO2 + H2O HCO3

− + H+

Enolase Mechanism

Entropic and Enthalpic Factors in Catalysis

Proximity and orientation effects Transition state

stabilization through preferential binding of transition state

Proximity and Orientation Effects

Enzymes Are Complementary to Transition State

Serine Protease Mechanism: Multiple Catalytic Mechanisms at Work

Structure of the Serine Protease Chymotrypsin

Serine Protease Substrate Specificity and Active-Site Pockets

Substrate specificity in serine proteases through active-site binding of side chain of amino acid residue adjacent to amide bond that will be cleaved.

Trypsin cleaves amide bond immediately C- terminal to basic amino acid residues.

Chymotrypsin cleaves amide bond immediately C-terminal to hydrophobic amino acid residues.

Serine Nucleophile in Serine Proteases

The Pre-Steady State in Chymotrypsin-Catalyzed Hydrolysis

of p-Nitrophenyl Acetate

E + S ES EP2 E + P2k2 k3

P1 H2Ok1

k-1

vo = kcat[E]t[S]/(KM + [S])Steady-state velocity, wherekcat = k2k3/(k2 + k3)KM = KSk3/(k2 + k3)

KS = k-1/k1

For chymotrypsin with ester substrates: k2 >> k3

Release of P1 faster than EP2 breaks down to E + P2

Serine Protease Mechanism

•Acid-base catalysis•Covalent catalysis•Proximity/orientation effects•Also (not depicted here) - electrostatic catalysis and transition state stabilization

Catalytic triad:

carboxylic acid,

The Oxyanion Hole In Serine Proteases

Role of oxyanion hole in serine protease mechanism:•Electrostatic catalysis•Preferential binding of transition state

Trypsin/Bovine Pancreatic Trypsin Inhibitor (BPTI) Complex

Trypsin-BPTI complex resembles tetrahedral transition state.

Transition State in Proline Racemase Reaction and Transition State Analogs

Proline racemase preferentially binds transition state, stabilizing it, and is potently inhibited by transition state analogs.

RNA-Based Catalysts (Ribozymes)

Cleavage of a Typical Pre-tRNA by Ribonuclease P

Ribonuclease P is a ribonucleoprotein (RNA- and protein-containing complex), and the catalytic component is RNA.

An even more complex example of an RNA- and protein-containing enzyme system is the ribosome. The central catalytic activity of the ribosome (peptide bond formation) is catalyzed by an RNA component.

tRNA substrate of ribonuclease P

Catalysis by the Intervening Sequence in Tetrahymena

Preribosomal RNA

RNA by itself without any protein can be catalytic.

Enzyme Regulation

Effect of Cooperative Substrate Binding on Enzyme Kinetics

Cooperative enzymes do not obey simple Michaelis-Menten kinetics.

Effect of Extreme Homoallostery

Homotropic allosteric regulation by substrate: S at one active site affects catalysis of S P at other sites on enzyme complex.

[S]c = critical substrate concentration

Extreme positive cooperativity depicted here

Heteroallosteric Control of an Enzyme

Heterotropic allosteric regulation: non-S effectors modulate catalysis of S P.

(positive allosteric effector)

(negative allosteric effector)

A Model for Enzyme Regulation: Aspartate Transcarbamoylase

(Aspartate Carbamoyltransferase) in Pyrimidine Synthesis

Aspartate Transcarbamoylase (ATCase)-Catalyzed Reaction

Feedback Inhibition of ATCase by CTP

Regulation of ATCase by ATP and CTP

ATP is a positive heterotropic allosteric effector of ATCase, while CTP is a negative heterotropic allosteric effector.

Detailed Structure of One Catalytic Subunit and Adjacent Regulatory

Subunit of ATCase

Quaternary Structure of ATCase in T State and R State

CTP and ATP bind at regulatory site, but CTP preferentially binds in T state, while ATP preferentially binds R state.

X-Ray Structure of Aspartate Transcarbamoylase

T State

R State

“top” view “side” view

“side” view

T State