title: Novel kinetic analysis of enzymatic dipeptide synthesis: effect of pH and substrates on thermolysin catalysis.
abstract: The point of maximum activity is specific to a particular substrate-enzyme system but may vary with different substrates and the same enzyme. The specificity of enzymes has, however, been generally reported only at their "optimal" pH. In this article, we introduce the Michaelis-Menten equation taking pH into account, and apply it to the pH-activity profile of the thermolysin-catalyzed dipeptide synthesis. It has been reported to date that the pH-activity profile of thermolysin follows a bell-shaped curve with a maximal activity at or near pH 7.0. The profiles obtained in this study, however, indicated that the optimal pH varied from 5.8 (for F-AspPheOMe) to 7.3 (for Z-ArgPheOMe), and the order of thermolysin activity was greatly dependent on the pH of reaction media. We have succeeded in evaluating the substrates-induced change of the dissociation states of the active site of thermolysin using the hydrophobicity of substrates. We have obtained apparent kinetic parameters which are independent of the pH of reaction media. The apparent specificity of thermolysin which were independent of pH of the reaction media was in order L-Leu > L-Asp > L-Arg > L-Ala > L-Gly > L-Val and Z > Boc = F at P1 and P2 positions, respectively.
authors: Department of Chemical Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555; Japan. muray@d5.dion.ne.jp
authors: Y
authors: Y
authors: Murakami
authors: K
authors: K
authors: Chiba
authors: T
authors: T
authors: Oda
authors: A
authors: A
authors: Hirata
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism*; D002384:Catalysis; D004151:Dipeptides / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity / Q000502:physiology; D013820:Thermolysin / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D002264:Carboxylic Acids; D004151:Dipeptides; D013820:Thermolysin