Pre-Steady-State Analysis of the Nucleoside Hydrolase of Trypanosoma vivax. Evidence for Half-of-the-Sites Reactivity and Rate-Limiting Product Release
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Abstract
The nucleoside hydrolase (NH) of the Trypanosoma vivax parasite catalyzes the hydrolysis of the N-glycosidic bond in ribonucleosides according to the following reaction: beta-purine (or pyrimidine) nucleoside + H(2)O --> purine (pyrimidine) base + ribose. The reaction follows a highly dissociative nucleophilic displacement reaction mechanism with a ribosyl oxocarbenium-like transition state. This paper describes the first pre-steady-state analysis of the conversion of a number of purine nucleosides. The NH exhibits burst kinetics and behaves with half-of-the-sites reactivity. The analysis suggests that the NH of T. vivax follows a complex multistep mechanism in which a common slow step different from the chemical hydrolysis is rate limiting. Stopped-flow fluorescence binding experiments with ribose indicate that a tightly bound enzyme-ribose complex accumulates during the enzymatic hydrolysis of the common purine nucleosides. This is caused by a slow isomerization between a tight and a loose enzyme-ribose complex forming the rate-limiting step on the reaction coordinate.
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