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• W2039372756 abstract "Trypanosoma cruzi S-adenosylmethionine decarboxylase (AdoMetDC) catalyzes the pyruvoyl-dependent decarboxylation of S-adenosylmethionine (AdoMet), which is an important step in the biosynthesis of polyamines. The time course of the AdoMetDC reaction under single-turnover conditions was measured to determine the rate of the slowest catalytic step up to and including decarboxylation. Analysis of this single-turnover data yields an apparent second-order rate constant for this reaction of 3300 M(-1) s(-1) in the presence of putrescine, which corresponds to a catalytic rate of >6 s(-1). This rate is minimally 100-fold faster than the steady-state rate suggesting that product release, which includes Schiff base hydrolysis, limits the overall reaction. AdoMetDC exhibits an inverse solvent isotope effect on the single-turnover 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 11 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 decarboxylated substrate, while little Schiff base species of either substrate or product was trapped with the C82A mutant. These data suggest that Cys-82 functions as a general acid/base to catalyze Schiff base formation and hydrolysis. The solvent isotope and pH effects are mirrored in single-turnover analysis of reactions without the putrescine activator, yielding an apparent second-order rate constant of 150 M(-1) s(-1). The presence of putrescine increases the single-turnover rate by 20-fold, while it has relatively little effect on the affinity of the enzyme for product. Therefore, putrescine likely activates the T. cruzi AdoMetDC enzyme by accelerating the rate of Schiff base exchange." @default.
• W2039372756 created "2016-06-24" @default.
• W2039372756 creator A5008866938 @default.
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• W2039372756 date "2000-03-01" @default.
• W2039372756 modified "2023-10-18" @default.
• W2039372756 title "Single-Turnover Kinetic Analysis ofTrypanosoma cruziS-Adenosylmethionine Decarboxylase†" @default.
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• W2039372756 doi "https://doi.org/10.1021/bi991493r" @default.
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