THE KINETIC MECHANISM OF PURINE NUCLEOSIDE PHOSPHORYLASE FROM MYCOBACTERIUM TUBERCULOSIS

Rodrigo Gay Ducati (INCT-TB PUCRS); Diógenes Santiago Santos (INCT-TB PUCRS); Luiz Augusto Basso (INCT-TB PUCRS)

Current epidemiological studies estimate that around one third of the world's population is infected with Mycobacterium tuberculosis, the aetiological agent of human tuberculosis (TB), and at risk of developing the active disease. Statistical data indicate the occurrence of 8 to 10 million new TB cases and 3 million deaths annually. Approximately 95% of TB cases occur in developing nations, where 98% of the world's TB death cases happen. The increasing prevalence of TB, the emergence of drug-resistant strains of the tubercle bacillus, and the devastating effect of co-infection with the human immunodeficiency virus have led to an urgent need for the development of new and more efficient antimycobacterial agents. An attractive approach for drug design is to develop selective enzyme inhibitors for essential metabolic pathways for the pathogen. Purine nucleoside phosphorylase from M. tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of TB. Here it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2'-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P_i) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P_i binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction. The results described here should be useful to the design of MtPNP inhibitors with potential action against M. tuberculosis.