PH dependence of the formation of dimeric alpha-chymotrypsin and its catalytic activity.

Abstract

The dimerization of alpha-chymotrypsin (alpha-CT) has been known to involve a specific interaction between the Tyr-146 alpha-carboxyl of one molecule and the His-57 imidazole, which is a member of the catalytic triad in the active site, of the other. This interaction determines the pH dependences of the dimerization constant and of the catalytic activities of the monomeric and dimeric enzymes. We compared the pKa values for catalytic activities with known pKa values for the dimerization constant in order to assign pKa values to residues of the enzyme. In the monomeric enzyme, the catalytic triad has a pKa value of 3.6 at the site between the Asp-102 carboxyl and His-57 N delta 1, and the Tyr-146 alpha-carboxyl has a pKa value of 4.6. In the dimeric enzyme, the site between the Asp-102 carboxyl and His-57 N delta 1 has a pKa value of around 5.5 and the site between His-57 N epsilon 2 and the Tyr-146 alpha-carboxyl has a pKa value around 2.4. Protonation at the site between the Asp-102 carboxyl and His-57 N delta 1 reduced the catalytic activity of the dimeric enzyme for p-nitrophenyl propionate, indicating that the Asp-102 carboxyl plays an important role in the monomeric alpha-CT-catalyzed reactions.