Biochem E2: Proteases

Ser/Thr Proteases

nuc attack by Ser/Thr, trypsin

Cys Protease

nuc attack by Cys, apoptosis

Aspartate Proteases

Acid-base catalysis, HIV-1 protease

Metalloproteases

nuc attack by H20, membrane proteases

Catalytic Triad of Ser Proteases

Asp, His, Ser

Importance of catalytic triad

collaborate to catalyze the reaction, highly conserved

Step 1 of Serine Proteases

General base catalysis and nucleophilic attack to form tetrahedral intermediate. His acts as a general base to pull hydrogen from the Ser OH group to form a strong nucleophile

Step 2 of serine proteases

General acid catalysis (by His) aids breakdown of the tetrahedral intermediate to the acyl-enzyme intermediate

Step 3 of serine proteases

General base catalysis (by His) and nucleophilic attack (by water) to form tetrahedral intermediate

Step 4 of serine proteases

General acid catalysis (by His) aids breakdown of tetrahedral intermediate to the carboxyl product. Ser accepts hydrogen from His to reform active site

Identification of the nucleophilic serine residue

reacting the enzyme with DFP (mimics TS, binds active site tightly) to form an irreversibly inactivated covalent adduct, which is then mapped via peptide hydrolysis

oxyanion hole

stabilizes the unstable tetrahedral intermediate formed when the serine nucleophile attacks the peptide carbonyl carbon during proteolysis

What changes to zymogen after cleavage to become active?

conformational change, cleavage forms the oxyanion hole

Determining the contribution of catalytic triad residues

mutagenesis studies and compare to WT, single mutants and triple mutant all had same change on catalysis suggesting cooperation, since some triple mutant is still catalyzing, something else must be playing a role (led to identification of oxyanion hole)

Allosteric Regulation of Ser Proteases

Peptide binding creates functional oxyanion hole

Example of allosteric regulation of Ser Proteases

Initiation of the envelope-stress response in E. coli, DegS is regulated by binding of unfolded OMP proteins, under stress OMP gets trapped in periplasm, OMP binds the PDZ domain of DegS and allosterically activates it to cleave RseA