Classification of Proteases Family Example Carboxy Metal Protease

Classification of Proteases Family Example Carboxy. Metal Protease peptidase A Chymotrypsin Serine Trypsin Protease Mechanism Zn 2+ E 72 H 69 H 196 S 195 -OH 57 D 102 C 25 -S- Cysteine Protease Papain H 195 Aspartyl Protease Pepsin Renin D 215 H 2 O D 32 Specificity Inhibitor Nonpolar EDTA EGTA DFP Aromatic TLCK Basic TPCK Non- PCMB specific Leupeptin Nonspecific Pepstatin Juang RH (2004) BCbasics

Modification of Subtilisin and Its Activity Change Active Site Modification Triad: Ser His Asp No enzyme Subtilisin ● ● ● 10, 000, 000 Ser, His & Asp → Ala ○ ○ ○ Relative activity 1 His & Asp → Ala ● ○ ○ 4, 000 37, 000 Ser → Ala Asp → Ala ○ ● ● ○ 5, 000 330, 000 Asn 155 → Leu ● ● ● 10, 000 (Asn 155 stabilizes transition state) Adapted from Dressler & Potter (1991) Discovering Enzymes, p. 245

Ser 195 Chymotrypsin Trypsin Elastase Thrombin Plasmin Acetylcholinesterase – Gly – Asp – Ser – Gly – Pro – Leu – – Gly – Asp – Ser – Gly – Pro – Val – – Gly – Asp – Ser – Gly – Pro – Leu – – Gly – Asp – Ser – Gly – Pro – Phe – – Gly – Asp – Ser – Gly – Pro – Leu – – Gly – Glu – Ser – Ala – Gly – Ala – His 57 Chymotrypsin Trypsin Elastase Thrombin Plasmin Acetylcholinesterase – Val – Thr – Ala – His – Cys – Gly – – Val – Ser – Ala – Gly – His – Cys – Tyr – – Leu – Thr – Ala – His – Cys – Ile – – Leu – Thr – Ala – His – Cys – Leu – – – – His – – – – Asp 102 Serine Protease and Ach. E Chymotrypsin Trypsin Elastase Thrombin Plasmin Acetylcholinesterase – Thr – Ile – Asn – Asp – Ile – Thr – – Tyr – Leu – Asn – Asp – Ile – Met – – Ser – Lys – Gly – Asn – Asp – Ile – Ala – – Asn – Leu – Asp – Arg – Asp – Ile – Ala – – Phe – Thr – Arg – Lys – Asp – Ile – Ala – – – – Asp – – – – Adapted from Dressler & Potter (1991) Discovering Enzymes, p. 244

Ach. E Has Similar Catalytic Mechanism O CH 3 + CH 3–C–O–CH 2–N–CH 3 O- H C O CH 3–C OC H Ach. E Acylation↑ ↓Deacylation H Ach. E O CH 3–C–OH O- H C CH 3 + O–CH 2–N–CH 3 H 2 O O CH 3–C CH 3 + HO–CH 2–N–CH 3 H-O-H O C H Ach. E Adapted from Dressler & Potter (1991) Discovering Enzymes, p. 243

Different Enzymes Might Adopt Same Mechanism OC Hi, Everybody! ← Useful ↙ Amusing Sesame Triad Juang RH (2004) BCbasics

Convergent and Divergent Molecular O Evolution C C O Acetylcholinesterase C Ester bond Peptide bond O C C C Thyroglobulin N H Chymotrypsin hydrolyze acetylcholine Convergent evolution Asp--His--Ser Serine Protease Divergent evolution Trypsin Elastase Thrombin Plasmin Juang RH (2004) BCbasics