Pre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release.
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.
Department of Ultrastructure, Vlaams Interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium. avdemeul@vub.ac.be
An
A
Vandemeulebroucke
Wim
W
Versées
Stefan
S
De Vos
Els
E
Van Holsbeke
Jan
J
Steyaert
D000818:Animals; D001665:Binding Sites; D006151:Guanosine / Q000737:chemistry; D006868:Hydrolysis; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry*; D011684:Purine Nucleosides / Q000737:chemistry; D012266:Ribose / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013379:Substrate Specificity; D016689:Trypanosoma vivax / Q000201:enzymology*
D015800:Protozoan Proteins; D011684:Purine Nucleosides; D006151:Guanosine; D012266:Ribose; D009699:N-Glycosyl Hydrolases