|Title||Pre-steady-state analysis of the nucleoside hydrolase of Trypanosoma vivax. Evidence for half-of-the-sites reactivity and rate-limiting product release.|
|Publication Type||Journal Article|
|Year of Publication||2003|
|Authors||Vandemeulebroucke, A., W. Versées, S. De Vos, E. Van Holsbeke, and J. Steyaert|
|Date Published||2003 Nov 11|
|Keywords||Animals, Binding Sites, Guanosine, Hydrolysis, Kinetics, N-Glycosyl Hydrolases, Protein Conformation, Protozoan Proteins, Purine Nucleosides, Ribose, Spectrometry, Fluorescence, Spectrophotometry, Substrate Specificity, Trypanosoma vivax|
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.