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Enzyme Mechanisms

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Page 1: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Enzyme Mechanisms

Page 2: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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.

Page 3: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Two Models for Enzyme-Substrate Interaction

Page 4: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Induced Conformational Change in Hexokinase

Page 5: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Coenzymes

Page 6: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Example of a Coenzyme Involved in Oxidation/Reduction Reactions

(nicotinamide adenine dinucleotide)

Hydride (H:-) transfer

Page 7: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Stereospecificity of Yeast Alcohol Dehydrogenase

pro-R

pro-S

ethanol acetaldehyde

Vennesland and Westheimer, 1950

pro-S

pro-R

Page 8: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Stereospecificity Conferred by an Enzyme

Page 9: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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)

Page 10: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Acid-Base Catalysis

Page 11: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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

Catalyzed

Page 12: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Example of Acid-Base Catalysis: Bovine Pancreatic RNase A

Page 13: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Covalent Catalysis: Nucleophiles and Electrophiles

Protonated

Page 14: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Example of Covalent Catalysis:Decarboxylation of Acetoacetate

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

Electrophilic “electron sink”

Page 15: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Example of Metal Ion Catalysis: Carboxypeptidase A

Page 16: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Example of Metal Ion Catalysis: Carbonic Anhydrase

Carbonic anhydrase catalyzes the reaction:CO2 + H2O HCO3

− + H+

Page 17: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Enolase Mechanism

Page 18: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Entropic and Enthalpic Factors in Catalysis

Proximity and orientation effects Transition state

stabilization through preferential binding of transition state

Page 19: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Proximity and Orientation Effects

Page 20: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Enzymes Are Complementary to Transition State

Page 21: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Serine Protease Mechanism: Multiple Catalytic Mechanisms at Work

Page 22: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Structure of the Serine Protease Chymotrypsin

Page 23: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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.

Page 24: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Serine Nucleophile in Serine Proteases

Page 25: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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

Page 26: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Serine Protease Mechanism

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

Catalytic triad:

Page 27: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 28: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 29: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 30: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 31: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 32: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

carboxylic acid,

Page 33: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 34: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

The Oxyanion Hole In Serine Proteases

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

Page 35: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Trypsin/Bovine Pancreatic Trypsin Inhibitor (BPTI) Complex

Trypsin-BPTI complex resembles tetrahedral transition state.

Page 36: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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.

Page 37: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

RNA-Based Catalysts (Ribozymes)

Page 38: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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

Page 39: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Catalysis by the Intervening Sequence in Tetrahymena

Preribosomal RNA

RNA by itself without any protein can be catalytic.

Page 40: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Enzyme Regulation

Page 41: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from
Page 42: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Effect of Cooperative Substrate Binding on Enzyme Kinetics

Cooperative enzymes do not obey simple Michaelis-Menten kinetics.

Page 43: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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

Page 44: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Heteroallosteric Control of an Enzyme

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

(positive allosteric effector)

(negative allosteric effector)

Page 45: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

A Model for Enzyme Regulation: Aspartate Transcarbamoylase

(Aspartate Carbamoyltransferase) in Pyrimidine Synthesis

Page 46: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Aspartate Transcarbamoylase (ATCase)-Catalyzed Reaction

Page 47: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Feedback Inhibition of ATCase by CTP

Page 48: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Regulation of ATCase by ATP and CTP

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

Page 49: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

Detailed Structure of One Catalytic Subunit and Adjacent Regulatory

Subunit of ATCase

Page 50: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

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.

Page 51: Enzyme Mechanisms. Types of Enzymes 1.Oxidoreductases catalyze oxidation-reduction reactions. 2.Transferases catalyze transfer of functional groups from

X-Ray Structure of Aspartate Transcarbamoylase

T State

R State

“top” view “side” view

“side” view

T State