Pre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release.

TitlePre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release.
Publication TypeJournal Article
Year of Publication2003
AuthorsVandemeulebroucke, A., W. Versées, S. De Vos, E. Van Holsbeke, and J. Steyaert
JournalBiochemistry
Volume42
Issue44
Pagination12902-8
Date Published2003 Nov 11
ISSN0006-2960
KeywordsAnimals, Binding Sites, Guanosine, Hydrolysis, Kinetics, N-Glycosyl Hydrolases, Protein Conformation, Protozoan Proteins, Purine Nucleosides, Ribose, Spectrometry, Fluorescence, Spectrophotometry, Substrate Specificity, Trypanosoma vivax
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.

DOI10.1021/bi0347914
Alternate JournalBiochemistry
PubMed ID14596604