Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei. Purification, specificity, and kinetic mechanism

Trypanosomes have no de novo purine biosynthesis and thus depend upon salvage pathways to obtain purines for their metabolic pathways and for the biosynthesis of nucleic acids. An inosine-adenosine-guanosine preferring nucleoside hydrolase (IAG-nucleoside hydrolase) from the African trypanosome Trypanosoma brucei brucei represents 0.2 percent of the soluble protein in this organism. The enzyme has been purified over 400-fold to>95 percent homogeneity from the bloodstream form of this parasite. IAG-nucleoside hydrolase is a dimer of Mr 36,000 subunits. The k cat/km for inosine, adenosine, and guanosine are 1.9 x 10(6), 1.2 x10(6), and 0.83 x 10(6) M-1 d-1, respectively. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The turnover rate for inosine at 30 degree C is 34 s-1. Pyrimidine nucleosides are poor substrates with k cat/Km values of approximately 10(3) M-1 s-1. Deoxynucleosides are also poor substrates with kcat/Km values near 10(2) M-1 s-1. AMP is not a detectable substrate and there is no measurable purine nucleoside phosphorylase activity. 3-Deazaadenosine, 7-deazaadenosine (tubercidin), and formycin B are competitive inhibitors with K is of 1.8, 59, and 13 M, respectively. The Km shows a slight dependence on pH with a pH optimum around 7. The V max/km data indicate there are two ionizable enzymatic groups, pKa 8.6, required for the formation of the Michaelis complex. The V max data indicate three ionizable groups in-volved in catalysis. Two essential groups exhibit pKa values of 8.8, and a third group with a pKa of 6.5 increases the V max an additional 10-fold. All three groups must be protonated for optimum catalytic activity.

Bibliographic Details

Main Author:	Parkin, D.W.
Format:	Journal Article biblioteca
Language:	English
Published:	Elsevier 1996-09
Subjects:	trypanosoma brucei, nucleosides, hydrolases,
Online Access:	https://hdl.handle.net/10568/32992 https://doi.org/10.1074/jbc.271.36.21713
Tags:	Add Tag No Tags, Be the first to tag this record!