Examination of the mechanism and energetic contribution of leaving group activation in the purine-specific nucleoside hydrolase from Trypanosoma vivax.

TitleExamination of the mechanism and energetic contribution of leaving group activation in the purine-specific nucleoside hydrolase from Trypanosoma vivax.
Publication TypeJournal Article
Year of Publication2007
AuthorsBarlow, J. N., and J. Steyaert
JournalBiochim Biophys Acta
Volume1774
Issue11
Pagination1451-61
Date Published2007 Nov
ISSN0006-3002
KeywordsAnimals, Binding Sites, Kinetics, N-Glycosyl Hydrolases, Thermodynamics, Trypanosoma vivax
Abstract

The mechanism and energetics of the purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) are examined by stopped-flow at low temperatures. TvNH is shown to follow an ordered uni-bi kinetic mechanism and high forward commitment with inosine as substrate (C(f) = 1.9 +/- 0.6). Measurement of partitioning of the Michaelis complex, which exists at negligible concentrations in the steady state, is achieved using a novel sequential-mixing stopped-flow method. A product burst is observed with p-nitrophenyl riboside (pNPR) in the pre-steady state, indicating that a step after chemistry rate determines k(cat). Comparison of the kinetics of inosine and pNPR turnover shows that the dominant energetic contribution towards catalysis in TvNH comes from ribosyl and water activation (11 kcal/mol); however, leaving group activation still makes a considerable (8 kcal/mol) contribution. A solvent isotope effect ((D2O)k = 1.7) on the chemistry transient tau1 with guanosine as substrate was observed. Therefore, the leaving group is unlikely to be protonated prior to N-glycosidic bond cleavage. We propose that leaving group protonation is, by itself, unlikely to account for the large energetic contribution of leaving group activation. Instead, we postulate that active site binding interactions to the purine leaving group are required for efficient ribosyl and/or water activation.

DOI10.1016/j.bbapap.2007.08.027
Alternate JournalBiochim. Biophys. Acta
PubMed ID17936095