Question

How does isoleucine 16 stabalize chymotrypsin active site? what would raising the pH well past 8 do?

Answer 1

Chymotrypsinogen is an inactive form of chymotrypsin. It is composed of 245 amino acids. A peptide bond between arginine 15 and isoleucine 16 is cleaved by trypsin to form pi-chymotrypsin. Pi-chymotrypsin acts on other pi chymotrypsin molecules to form alpha chymotrypsin. In alpha chymotrypsin, to dipeptides are removed to form three resulting chains A, B and C which are then linked by inter-chain disulphide bonds. A chain has amino acids 1-13, B chain has amino acids 16-146 and C chain has amino acids 149-245.

The amino terminal of the newly formed isoleucine 16 will then turn inward upon trypsin cleavage. It will form an ionic bond with aspartate 194. This aspartate is present in the interior of chymotrypsin. The protonation of this amino group of isoleucine is required for formation of active chymotrypsin. Subsequent to this ionic bond formation, Methionine 192 moves to the surface of enzyme from its deeply buried position. Residues 187 and 193 get extended. These conformational changes cause formation of substrate specificity site for aromatic and bulky amino groups. Hydrogen bonds form between negatively charged carbonyl oxygen atom in substrate and tow NH groups present in enzyme. These hydrogen bonds stabilize the tetrahedral transition state formed during catalysis. Thus, isoleucine 16 plays an important role in forming the active site of chymotrypsin.

The optimum pH of chymotrypsin is around 8. The amino terminal of Isoleucine 16 is required to be properly protonated in active chymotrypsin. A properly protonated amino terminus of this amino acid will position Asp 194, when forming an ionic bond, for optimal substrate binding backbone. This alpha amino group has a pKa value of nearly 9-10. Hence, if pH increases above 8 (or more than 9), the there is an increase in Km. At this increased Km, the enzyme will require more substrate to reach Vmax or maximum velocity. Hence, the activity of chymotrypsin will decrease at pH above 8.