Catalytic Mechanisms. Objective To understand how enzymes work at the molecular level. Ultimately requires total structure determination, but can learn.

Catalytic Mechanisms

Objective

To understand how enzymes work at the molecular

level.

Ultimately requires total structure determination, but can learn much through biochemical

analysis.

To Be Explained

• Specificity– For specific substrates– Amino acids residues involved

• Catalysis– Mechanisms– Amino acids involved/Specific role(s)

Enzyme Binding Sites

• Active Site:– Substrate Binding Site + Catalytic Site

• Regulatory Site: – a second binding site, – Binding by regulatory molecule affects the active site

• alter the efficiency of catalysis • improve or inhibit

General Characteristics

• Three dimensional space• Occupies small part of enzyme volume• Clefts or crevices

• Ligands (substrate or effector) bound by multiple weak interactions

• Specificity depends on precise arrangement of atoms in active site

Models

Lock and Key Induced Fit

Identification and Characterization of Active

Site

• Structure: size, shape, charges, etc.

• Composition: identify amino acids involved in binding and catalysis.

Binding or Positioning Site(Trypsin)

NH CH C NH

O

N C

complementary binding or posit ioning site

"SPECI FI CI TY"_

+

arginine or lysine

"long + side chain"

H2O

Binding or Positioning Site(Chymotrypsin)

NH CH C NH

O

N C

"aromatic side chain"

"SPECI FI CI TY"

complementary binding or posit ioning site

phenylalaninetyrosinetryptophan

Hydrophobic Pocket

H2O

O

Catalytic Site(e.g. Chymotrypsin)

NH CH C NH

O

N C

catalytic sitecomplementary

"CATALYSI S"

H2O

O

Probing the Structure of the Active Site

Model Substrates

Model Substrates(Chymotrypsin)

H2O(ROH)

NH CH CN

R

NH

O

C

acyl transfer to H2Oaromaticside chain

peptide bond

Peptide Chain?

All Good Substrates!

H3N CH C NH

O

C

R

NH CH CH3N

R

NH2

O

(or -OCH3)

or

H3N CH C

R

NH2

O

(or -OCH3)

or

-amino group?

Good Substrate!

H2C C NH2

O

R (OCH3)

Side Chain Substitutions

Good Substrates

Cyclohexyl t-butyl-

CH3

CH3

CH3

ConclusionBulky Hydrophobic Binding Site

CH C X

O

Y

"Hydrophobic Acyl Group Transferase"

= hydrophobic posit ioning group

X,Y = various

Probing the Structure of the Active Site

Competitive Inhibitors

Arginase

H2N

C

NH

(CH2)3

CH COOH3N

NH2H2O

NH3

(CH2)3

CH COOH3N

H2N

C

O

NH2

+

+

-+

ureaornithinearginine

+ -

+

Good Competitive Inhibitors

NH3

NH

NH3

(CH2)3

CH COOH3N

NH3

(CH2)4

CH COOH3N

O

(CH2)2

CH COOH3N

CH

NH2

-+

(

ornithine

(+

-

++

+

-

(

canavaninelysine

+

Poor Competitive Inhibitors

All Three Charged Groups are Important

NH3

(CH2)3

CH2H3N

NH3

(CH2)3

H2C COO

CH3

(CH2)3

CH COOH3N+ -

++

+ -

a-aminovaleric acid putrescine

(l,4-diaminobutane)4-aminovaler ic acid

ConclusionActive Site Structure of Arginase

+-

-bindingsite

catalytic site

Identifying Active Site Amino Acid Residues

• Covalent modification of residues– Inactivation of enzyme

• Site directed mutagenesis– Inactivation of enzyme

Mechanisms of Catalysis

• Acid-base catalysis

• Covalent catalysis

• Metal ion catalysis

• Proximity and orientation effects

• Preferential binding (stabilization) of the transition state

Acid-Base Catalysis

Addition or removal of a proton by side chains

General Acids and Bases

Acid-Base Catalysis

Keto-Enol Tautomerization

R C CH3

O

R C

OH

CH2

Ketone Enol

Uncatalyzed Reaction

General Acid Catalysis

General Base Catalysis

Figure 11-10

Ribonuclease A

N

N

O

O

OH

O

O

CH2

O

PO O

O

N

N

N

N

O

NH2

OH

CH2OP

O

O

O

PO O

O

Adenosine

UridineRibonuclease A

Figure 11-10 part 1

Mechanism of RNase A

Figure 11-10 part 2

Mechanism of RNase A

Covalent Catalysis(Nucleophilic catalysis)

(Principle)

Involves a transient covalent bond between the enzyme and the substrate

Usually by the nucleophilic attack of the substrate by the enzyme

Covalent Catalysis(Principle)

SlowH2O + A–B ——> AOH + BH

A-B + E-H ——> E-A + BHE-A + H2O ——> A-OH + E-H

Fast

NOTE: New Reaction Pathway

Covelent Catalysis

The Schiff Base

Metal Ion Catalysis

• Charge stabilization

• Water ionization

• Charge shielding

• Metalloenzymes: tightly bound metal ions– Catalytically essential– Fe2+, Fe3+, Cu2+, Mn2+, and Co2+

• Metal-activated enzymes: loosely bound metal ions (from solution or with substrate)– Structural metal ions: – Na+, K+, and Ca2+

• Both: Mg2+ and Zn2+

Metal Ion Catalysis

Carbonic Anhydrase

Carbonic Anhydrase

Proximity and Orientation Effects

Rate of a reaction depends

on:

• Number of collisions• Energy of molecules• Orientation of molecules• Reaction pathway (transition state)

Proximity

V = k[A][B]

[A] and [B] = ~13M on enzyme surface

Page 336

Biomolecular Reaction of Imidazole with p-Nitrophenylacetate

(Intermolecular)

Page 336

Intramolecular Reaction of Imidazole with p-Nitrophenylacetate

(Intramolecular)

Intramolecular Rate = 24x Intermolecular Rate

Orientation

A BBA

C

Figure 11-14

Geometry of an SN2 Reaction

Preferrential Binding of Reaction Intermediate

• Stabilize Transition State– Electrostatic stabilization of developing charge

– Relief of induced bond angle strain

– Enhancement of weak interactions between enzyme and intermediate.

Page 338

Steric Strain in Organic Reactions

Reaction Rate: R=CH3 is 315x vs R=H

Figure 11-15

Effect of PreferentialTransition State Binding

Transition State Analogs

Powerful Enzyme Inhibitors

Page 339

Proline Racemase(planar transition state)

Page 339

Transition State Analogs of Proline

Binding = 160x versus Proline

Serine Proteases

ChymotrypsinTrypsinElastase

etc.

Convergent Evolution

Substrate Specificity

Mechanism of Chymotrypsin

CT CH2 OH O2N O C CH3

O

CT CH2 O C CH3

O

CT CH2 OH

OO2N

CT CH2 O C CH3

O

HO C CH3

O

"rate limiting"

++ H2O

+fast

+

fast

p-nitrophenolate

X-Ray Structure of Bovine Trypsin(Ribbon Diagram)

Figure 11-26

Active Site Residues of Chymotrypsin(Catalytic Triad)

Catalytic Mechanism of the Serine Proteases

Catalytic Triad

Figure 11-29 part 2

Catalytic Mechanism of the Serine Proteases

Catalytic Mechanism of the Serine

Proteases

Catalytic Mechanism of the Serine Proteases

Catalytic Mechanism of the Serine Proteases

Catalytic Mechanism of the Serine

Proteases

Catalytic Mechanism of the Serine

Proteases

Figure 11-30a

Transition State Stabilization in the Serine Proteases

Figure 11-30b

Transition State Stabilization in the Serine Proteases

Mechanism of Chymotrypsin

CT CH2 OH O2N O C CH3

O

CT CH2 O C CH3

O

CT CH2 OH

OO2N

CT CH2 O C CH3

O

HO C CH3

O

"rate limiting"

++ H2O

+fast

+

fast

p-nitrophenolate

New Reaction Pathway(versus uncatalyzed reaction)