Kinetics of Woodward's Reagent K hydrolysis and reaction with staphylococcal nuclease.

The kinetics of the hydrolysis of Woodward’s Reagent K has been studied as a function of pH in aqueous solution. Rate constants for the reagent transformation have been combined with rate data for loss of staphylococcal nuclease activity to provide the mechanism for the protein modification. The reactive species for derivatization of the enzyme is the intermediate ketoketenimine. The pH dependence of the rate of the second order reaction between this intermediate and the nuclease yielded a pKBPp of 5.22, which is attributed to a carboxyl group or groups of the enzyme. limited to the alkaline pH region and therefore participation by a carboxylate anion would not be evident. Thus, it seemed worthwhile to attempt the modification of S. nuclease with Woodward’s reagent K in an effort to gain further insights into the role of carboxyl groups in this enzyme. We have found that WRK effects a rapid inactivation of S. nuclease. In the course of this study, we have measured the rate of hydrolytic decomposition of WRK in aqueous solution under conditions identical with those employed for the modification experiments. These rate constants allow the determination of the microscopic rat.e constants of modification of the protein when combined with the time course of inactivation.