Chapter 9:Enzymes:

Catalytic Strategies

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition

Types of Catalysis

1. Covalent2. Acid-Base

1. General acid-base (Bronstead acid or base, HA or A-)

2. Specific acid-base (solvent, e.g. water, H+ or OH-)

3. Metal ion4. Binding Effects

1. Approximation (proximity)2. Transition state stabilization

Proteases or Peptidases

These enzymes cleave peptide bonds.Remember that a peptide bond is an amide bond and an amide is the least reactive carboxylic acid derivative. It is very resistant to hydrolysis.Enzymes catalyze this reaction in milliseconds.

protease

ChymotrypsinChymotrypsin is an intestinal protease that Recognizes and binds non-polar sidechains, primarily aromatic sidechains: Phe, Tyr, Trp

Cleaves

Cleaves slower

A Chromogenic Substrate

Esterase activityCleaves

Yellow Color

Covalent Inhibition at Ser-195 of Chymotrypsin

Mechanism

Covalent catalysis – two steps

Fast Slow

A covalentintermediate

Active Site Titration

Acylation is rapid

Deacylation is slow

Catalytic Triad in Chymotrypsin

The cataytic triad makes Ser-195 the only acidicSer in chymotrypsin. The developing alkoxide is an excellent nucleophile.

Mechanism

Attack at the Peptide bond

Stabilization of the tetrahedral intermediate

The O- forms ion-dipoles with two peptide N-Hhydrogens in the oxyanion hole.

Reform carbonyl and release N-terminus

Dissociation of the

N-Terminus

New substrate

(water) enters

Stabilization of the tetrahedral intermediate

Again, the O- forms ion-dipoles with twopeptide N-H hydrogens in the oxyanion hole.

Reform carbonyl and release C-terminus

Dissociation of the

C-Terminus

Catalytic triad:Asp 102…His 57…Ser 195

Hydrophobic binding

pocket in chymotrypsin

Binding in other seryl enzymes

Papain – A Cysteine Protease

A Cysteine Protease with Substrate

Renin - An Aspartyl Protease

An Aspartyl Protease with Substrates

Pepsin - An Aspartyl Protease

A Typical Mechanism

O

CH2-C-O-

O

H-O-C-CH2

O

H

H

CH2-C-O-

O

H-O-C-CH2

O

C

R

O NH-R'

Asp32pKa 2.9 Asp215

pKa 4.5

Note the perturbed pKa values. Sidechain pKa of Asp is 3.86

Substrates in

Pepsin - Mechanism

OH

H

CH2-C-O-

O

H-O-C-CH2

O

C

R

O NH-R'

HO

CH2-C-O-H

O

-O-C-CH2

O

C

R

HO NH-R'

Tetrahedral intermediate

General acid & base catalysis

Pepsin - Mechanism

HO

CH2-C-O-

O

H-O-C-CH2

O

C

R

H -O NH-R'

HO

CH2-C-O-H

O

-O-C-CH2

O

C

R

ONH2 -R'

H on Asp32 moves to Asp215 followed by general acid & base catalysis

Peptide bond cleavage

Pepsin - Mechanism

HO

CH2-C-O-

O

H-O-C-CH2

O

C

R

ONH2 -R'

CH2-C-O-

O

H-O-C-CH2

O

H on Asp32 moves to Asp215 and products leave the active site

Enzyme is ready for substrate

A Dimeric Aspartyl Protease Flaps/trap doors to retain substrate

HIV Protease

Thermolysin – A Metalloprotease

A Metalloprotease with Substrate

Carbonic Anhydrase, a Zn++ enzyme

Carbonic Anhydrase reaction

Carbonic Anhydrase MechanismProton release

CO2 enters

HO- attacks CO2

HCO3-

released. H2Oenters

His64 assists in H+ removal

His64 Participation

Carbonic Anhydrase pH – Rate Profile

pKa of Zinc bound HOHchanges from 15.7 to ~7

Restriction EnzymesThese enzymes are endonucleases that cleave foreign DNA. They “restrict” invasion by foreign DNA by destroying it. They cleave at specific base sequences (recognition

sites). Host DNA with the same sequence is protected by methylation.These are Class 3 enzymes, hydrolases, EC 3.x.x.x).

Restriction EnzymesThere are three types of restriction enzymes: I, II & III. Types I and III require ATP for hydrolysis.Type II does not and Type II enzymes are the ones used for cloning and sequencing DNA.Hydrolysis by Type II enzymes generates a 3’ OH and

5’ phosphate.

CleavageAttack

Inversion of Configuration

Here one oxygen has been replaced with sulfur in order to determine the stereochemistry of reaction, i.e. inversion vs retention of configuration.

Mg++ needed by the Type II enzyme

A segment of DNA

Cleavage site

Restriction sites are Palindromes

H-Bonding of enzyme-DNA site

Methylation prevents H-

bonding with DNA substrate

Nucleoside Monophosphate (NMP) kinases

Adenylate kinase: Mg++

ATP + AMP < == > 2 ADP

Guanylate kinase: Mg++

ATP + GMP < == > ADP + GDP

NMP kinase reaction

ATP:Mg++ Complexes

Most enzymes that require ATP, actually require ATP:Mg++ as substrate and will not use ATP alone.

Kinases are of this type.

Isomeric forms

Hexacoordinate Mg++

Additional conformational change occurs after NMPbinds.

P Loop

Conserved G- X-X-X-X-G-K

X

X

X

X

End of Chapter 9

Copyright © 2007 by W. H. Freeman and Company

Berg • Tymoczko • Stryer

BiochemistrySixth Edition