Purine nucleoside phosphorylase from Mycobacterium tuberculosis. Analysis of inhibition by a transition-state analogue and dissection by parts

Purine salvage pathways are predicted to be present from the genome sequenc e of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a pr esumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expre ssed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Esche richia coli enzyme. Immucillin-H is a rationally designed analogue of the t ransition state that has been shown to be a potent inhibitor of mammalian P NP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K-i of 2.2 nM) followed by an overall dissociation constant (K-i*) of 28 pM, yielding a K-m/K-i* valu e of 10(6). Time-dependent tight binding of the inhibitor occurs with a rat e of 0.1 s(-1), while relaxation of the complex is slower at 1.4 x 10(-3) s (-1). The pH dependence of the K-i value of immucillin-H to the M. tubercul osis PNP suggests that the inhibitor binds as the neutral, unprotonated for m that is subsequently protonated to generate the tight-binding species. Th e M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. An alysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K-is of 0.3 9 muM) while the 1,4dideoxy-1,4-iminoribitol binds weakly (K-is of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypox anthine and iminoribitol (beta = 13). However, the covalent attachment of t hese two components in immucillin-H increases equilibrium binding affinity by a factor of > 14 000 (28 pM vs 0.39 muM) compared to 9-deazahypoxanthine alone, and by a factor of > 10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. t uberculosis PNP inhibition by immucillin-H and by its component parts is re ported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].