Purine-specific nucleoside N-ribohydrolase from Trypanosoma brucei brucei. Purification, specificity, and kinetic mechanism
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Journal of Biological Chemistry;271(36): 21713-21719
Permanent link to cite or share this item: http://hdl.handle.net/10568/32992
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.