L14/C6: Enzymatic Reactions

Biochemistry I

Proteases (peptidases)

Enzymes that catalyze the hydrolysis of peptide bonds, breaking proteins or peptides into smaller fragments

Exopeptidases

Cleave peptide bonds near the N- or C- terminus of polypeptides

Endopeptidases

Cleave internal peptide bonds within polypeptide chains

Four Major Mechanistic Categories

1. Aspartyl proteases

2. Metalloproteases

3. Serine proteases

4. Cysteine proteases

Aspartyl Proteases (ex Pepsin, HIV protease)

Catalytic Asp residues activate water for nucleophilic attack (acid-base catalysis)

Metalloproteases (ex Thermolysin, matrix metalloproteinases)

A metal ion (usually Zn²⁺) facilitates water activation and stabilizes the transition state

Serine proteases (Chymotrypsin, trypsin)

Catalytic Ser acts a nucleophile, forming a covalent acyl-enzyme intermediate

Cysteine proteases (ex Papain, casoases)

Catalytic Cys acts as a nucleophile, forming a covalent thioester intermediate

The Catalytic Triad

• A reactive Ser is the first (key) catalytic residue in Ser proteases

• A His residue is a second catalytic residue in Ser proteases

• An Asp is the third residue in the catalytic triad

• Residues are oriented in a very precise 3D orientations, in very specific distances; not the same sequence positions

Nerve Agents (Poisons)

• AChE is a serine esterase found at cholinergic synapses- it is the off switch for nerve impulse

• Parathion, malathion more toxic to insects than humans

• DIPF and sarin used as nerve gases

Protease

An enzyme that catalyzes the hydrolytic cleavage of peptide bonds

Ex: bovine pancreatic chymotrypsin

Chymotrypsin Mechanism Phase 1

Acylation Phase

• peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme

Chymotrypsin Mechanism Phase 2

Deacylation phase

• the ester linkage is hydrolyzed and the nonacylated enzyme is regenerated

The Chymotrypsin Reaction: Serine Protease

• Ser reside that acts as the nucleophile

• acylation phase nucleophile is the oxygen of Ser195

The Chymotrypsin Reaction: Catalytic Triad

• Hydrogen bonding network

• In chymotrypsin, Ser195 is linked to His57 and Asp102