Biochemistry Sixth Edition Berg • Tymoczko • Stryer Chapter 9: Enzymes: Catalytic Strategies Copyright © 2007 by W. H. Freeman and Company

Types of Catalysis Covalent Acid-Base General acid-base (Bronstead acid or base, HA or A-) Specific acid-base (solvent, e.g. water, H+ or OH-) 3. Metal ion 4. 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

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

A Chromogenic Substrate Cleaves Esterase activity Yellow Color

Covalent Inhibition at Ser-195 of Chymotrypsin

Mechanism Covalent catalysis – two steps Fast Slow A covalent intermediate

Active Site Titration Deacylation is slow Acylation is rapid

Catalytic Triad in Chymotrypsin The cataytic triad makes Ser-195 the only acidic Ser 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-H hydrogens 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 two peptide 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 Substrates in Note the perturbed pKa values. Sidechain pKa of Asp is 3.86

Pepsin - Mechanism Tetrahedral intermediate General acid & base catalysis Tetrahedral intermediate

Pepsin - Mechanism H on Asp32 moves to Asp215 followed by Peptide bond general acid & base catalysis Peptide bond cleavage

Pepsin - Mechanism H on Asp32 moves to Enzyme is ready 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 Mechanism Proton release His64 assists in H+ removal HCO3- released. H2O enters CO2 enters HO- attacks CO2

His64 Participation

Carbonic Anhydrase pH – Rate Profile pKa of Zinc bound HOH changes from 15.7 to ~7

Restriction Enzymes These 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 Enzymes There 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. Cleavage Attack

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 Cleavage site A segment of DNA

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: 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 NMP binds.

P Loop X X X X Conserved G- X-X-X-X-G-K

Biochemistry Sixth Edition Berg • Tymoczko • Stryer End of Chapter 9 Copyright © 2007 by W. H. Freeman and Company