title: Examination of the mechanism and energetic contribution of leaving group activation in the purine-specific nucleoside hydrolase from 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.
authors.affiliation: Department of Molecular and Cellular Interactions, VIB, Free University of Brussels, Pleinlaan 2, 1050 Brussel, Belgium. jbarlow@vub.ac.be
authors.forename: John N
authors.initials: JN
authors.lastname: Barlow
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
mesh_terms: D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry*; D013816:Thermodynamics; D016689:Trypanosoma vivax / Q000201:enzymology*
chemical_list: D009699:N-Glycosyl Hydrolases; C030681:purine nucleosidase