Purine-specific Nucleoside N-Ribohydrolase from Trypanosoma brucei brucei

Abstract

Trypanosomes 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) represents ~0.2% of the soluble protein of Trypanosoma brucei. The enzyme was purified >400-fold to >95% homogeneity from the bloodstream form and shown to be a dimer of Mr 36000 subunits. The kcat/Km for inosine, adenosine and guanosine were 1.9x106, 1.2x108 and 0.83x106 smallcap~M-1smallcap~S-1, respectively. The kinetic mechanism with inosine as substrate was rapid equilibrium with random product release. The turnover rate for inosine at 30 degrees C was 34 smallcap~S-1. Pyrimidine nucleosides were poor substrates with kcat/Km values of ~103 smallcap~M-1smallcap~S-1. Deoxynucleosides were also poor substrates with kcat/Km values near 102 smallcap~M-1smallcap~S-1. AMP was not a detectable substrate and there was no measurable purine nucleoside phosphorylase activity. 3-Deazaadenosine, 7-deazaadenosine (tubercidin) and formycin B were competitive inhibitors with Kis of 1.8, 59 and 13 micro smallcap~M, respectively. The Km showed a pH optimum of around 7. The Vmax/Km data indicated that there are 2 ionizable enzymatic groups, pKa 8.6, required for the formation of the Michaelis complex. The Vmax data indicated 3 ionizable groups involved in catalysis. Two essential groups exhibited pKa values of 8.8 and a third group with a pKa of 6.5 increased the Vmax an additional 10-fold. All 3 groups must be protonated for optimum catalytic activity.