Single-turnover kinetic analysis of Trypanosoma cruzi S-adenosylmethioninedecarboxylase

Trypanosoma cruzi S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes t he pyruvoyl-dependent decarboxylation of S-adenosylmethionine (AdoMet), whi ch is an important step in the biosynthesis of polyamines, The time course of the AdoMetDC reaction under single-turnover conditions was measured to d etermine the rate of the slowest catalytic step up to and including decarbo xylation. Analysis of this single-turnover data yields an apparent second-o rder rate constant for this reaction of 3300 M-1 s(-1) in the presence of p utrescine, which corresponds to a catalytic rate of >6 s(-1). This rate is minimally 100-fold faster than the steady-state rate suggesting that produc t release, which includes Schiff base hydrolysis, limits the overall reacti on. AdoMetDC exhibits an inverse solvent isotope effect on the single-turno ver kinetics, and the pH profile predicts a pK(a) of 8.9 for the basic limb . These results are consistent with a Cys residue functioning as a general acid in the rate-determining step of the single-turnover reaction. Mutation of Cys-82 to Ala reduces the rate of the single turnover reaction to II M- 1 s(-1) in the presence of putrescine. Further, a solvent isotope effect is not observed for the mutant enzyme. Reduction of the wild-type enzyme with cyanoborohydride traps the Schiff base between the enzyme and decarboxylat ed substrate, while little Schiff base species of either substrate or produ ct was trapped with the C82A mutant, These data suggest that Cys-82 functio ns as a general acid/base to catalyze Schiff base formation and hydrolysis. The solvent isotope and pH effects are mirrored in single-turnover analysi s of reactions without the putrescine activator, yielding an apparent secon d-order rate constant of 150 M-1 s(-1). The presence of putrescine increase s the single-turnover rate by 20-fold, while it has relatively little effec t on the affinity of the enzyme for product. Therefore, putrescine likely a ctivates the T. cruzi AdoMetDC enzyme by accelerating the rate of Schiff ba se exchange.