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