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 similar to 0.2% of the soluble protein in this organism. The enzyme has been purified over 400-fold to >95% homogeneity from the bloodstream form of this parasite. IAG-nucleoside hydrolase is a dimer of M(r) 36,000 subunits. The k(cat)/K-m for inosine, adenosine, and guanosine are 1.9 x 10(6), 1.2 x 10(6), and 0.83 x 10(6) M(-1) s(-1), respectively. The kinetic mechanism with inosine as substrate is rapid equilibrium with random product release. The turnover rate for inosine at 30 degrees C is 34 s(-1). Pyrimidine nucleosides are poor substrates with k(cat)/K-m values of approximately 10(3) M(-1) s(-1). Deoxynucleosides are also poor substrates with k(cat)/K-m 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 mu M, respectively. The K-m shows a slight dependence on pH with a pH optimum around 7. The V-max/K-m data indicate there are two ionizable enzymatic groups, pK(a) 8.6, required for the formation of the Michaelis complex. The V-max data indicate three ionizable groups involved in catalysis. Two essential groups exhibit pK(a) values of 8.8, and a third group with a pK(a) of 6.5 increases the V-max an additional 10-fold. All three groups must be protonated for optimum catalytic activity.