1. Chymotrypsin Is Activated by Proteolysis Adapted from Campbell (1999) Biochemistry (3d) p.179 245 R15-I16 Chymotrypsinogen (inactive) p -Chymotrypsin (active) S14-R15T147-N148 Trypsin a -Chymotrypsinogen (active) p -Chymotrypsin I16L13A149Y146 Disulfide bonds

2. Charge Relay in Active Site Ser 195 His 57 Asp 102 H–O–CH 2 O C–O - = Active Ser H–N N CC C H H CH 2 Ser 195 His 57 Asp 102 - O–CH 2 O C–O–H = N N–H CC C H H CH 2 Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158

3. pH Influences Chymotrypsin Activity 5 6 7 8 9 10 11 pH Relative Activity Adapted from Dressler & Potter (1991) Discovering Enzymes, p.162

4. pH Influences Net Charge of Protein Juang RH (2004) BCbasics + Net Charge of a Protein Buffer pH Isoelectric point, pI - 3 4 5 6 7 8 9 10 0 +

5. Imidazole on Histidine Is Affected by pH H–N N C C C H H H+H+ pH 6pH 7 + H–N N–H C C C H H Inactive + Ser 195 His 57 Asp 102 H–O–CH 2 O C–O - = H–N N–H CC-H C CH 2 H Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.158 Adapted from Dressler & Potter (2000) Discovering Enzymes, p.163

6. Chymotrypsin Produces New Ile16 N-Terminal I16L13Y146 Asp 194 –CH 2 COO - Ile 16 NH 2 – Ile 16 + NH 3 – 5 6 7 8 9 10 11 pH Relative activity pH 9 pH 10 pKa Adapted from Dressler & Potter (1991) Discovering Enzymes, p.165 New NH 2 -terminus

7. New Ile16 N-Terminal Stabilizes Asp194 Asp 102 His 57Ser 195 Asp 194 Gly 193 Ile 16 + NH 3 Catalytic Triad Adapted from Dressler & Potter (1991) Discovering Enzymes, p.206 Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.112

8. O (CH 3 ) 2 CH–O– P –O–CH(CH 3 ) 2 F = Chymotrypsin Ser195 Inhibited by DIFP Diisopropyl-fluorophosphate (DIFP) Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167 O - …H CH 2 Ser 195 O (CH 3 ) 2 CH–O– P –O–CH(CH 3 ) 2 = O CH 2 Ser 195 XX

9. Addition of Substrate Blocks DIFP Inhibition Reaction time Percent Inhibition of activity (%) 100 50 0 No substrate Add substrate S + DIFP + DIFP & substrate Adapted from Dressler & Potter (1991) Discovering Enzymes, p.167 XX

10. Chymotrypsin Also Catalyzes Acetate O -C N- H O -C O- Peptide bond Ester bond O CH 3 –C–O– –NO 2 Nitrophenol acetate HO– –NO 2 O CH 3 –C–OH Hartley & Kilby Chymotrypsin+ H 2 O Nitrophenol Acetate No acetate was detected at early stage Adapted from Dressler & Potter (1991) Discovering Enzymes, p.168

11. O -CO -C Time (sec) Nitrophenol Two-Stage Catalysis of Chymotrypsin O CH 3 –C–O– –NO 2 Nitrophenol acetate OCOC O CH 3 –C HO– –NO 2 + H 2 O O-H C CH 3 COOH Kinetics of reaction Two-phase reaction Acylation Deacylation (slow step) Adapted from Dressler & Potter (1991) Discovering Enzymes, p.169

12. Extra Negative Charge Was Neutralized O -C N- H O -C-OH NH 2 - -C-C-N-C-C-N-C-C-N- H H E + S Adapted from Dressler & Potter (1991) Discovering Enzymes, p.179 O - -C N- HO H O - -C N- HO H

13. Active Site Stabilizes Transition State Asp 102 His 57 Met 192 Gly 193 Asp 194Ser 195 Cys 191 Catalytic Triad Thr 219 Ser 218 Gly 216 Ser 217 Trp 215 Ser 214 Cys 220 Specificity Site Active Site Adapted from Dressler & Potter (1991) Discovering Enzymes, p.197