43869-G4
Kinetic Isotope Effects on the NAD+ Synthetase-Catalyzed Reaction
Current estimates indicate that one third of the worldÕs population-more than 2 billion people are asymptomatically infected with M. tuberculosis. Moreover, there are ten million new cases of active TB every year, resulting in two million deaths attributed to TB annually. Many of those deaths arise due to the lack of any antitubercular treatment, however, a growing number are caused by drug resistant M. tuberculosis strains. To achieve a major reduction in global TB there is a need to develop chemotherapeutic treatment regimens that eradicate not only the active form of the disease, but also target the non-replicating organisms that characterize latent TB. Cofactors, such as NAD+, participate in critical roles in most aspects of metabolism and their biosynthesis offers a rich source of potential drug targets. NAD+ metabolism comprises a complex network of different de novo biosynthetic and recycling pathways that differ, sometimes drastically, among organisms. NAD+ biosynthesis in M. tuberculosis is dependent on NAD+ synthetaseÕs activity. This enzyme has been classified as essential in M. tuberculosis by genome-wide transposon-mutagenesis. Mutants of the de novo biosynthetic pathway rapidly lost viability in the absence of nicotinamide and nicotinic acid pointing to the essentiality of NAD+ and of NAD+ synthetase for maintaining cellular viability in M. tuberculosis. NAD+ metabolism in humans and yeast contains more recycling pathways than that in M. tuberculosis. Two of these pathways are NAD+ synthetase independent. We have determined the steady-state rate constants for the M. tuberculosis NAD+ synthetase NAD+ synthetaseTB) catalyzed reactions using exogenous ammonia and using the ammonia produced by the hydrolysis of L-glutamine. The turnover number for the reaction utilizing ammonia is at least 5-6 fold faster, considering the difference in ionic strengths, than for the reaction utilizing L-glutamine. A rate limiting step involved in glutamine hydrolysis and/or ammonia transport could account for this difference. The Km for glutamine is not affected by the presence of the substrates of the synthetase domain. The degree of activation of the glutaminase activity by the synthetase substrate is an indication of the efficiency of the enzyme to minimize wasteful glutamine hydrolysis. A 179 fold activation of the glutaminase turnover number was measured for the reaction catalyzed by NAD+ synthetaseTB in the presence of both synthetase substrates. This suggests that the glutaminase domain undergoes a conformational change from an inactive to an active conformation upon binding of both NaAD+ and ATP to the synthetase domain. The stoichiometry between the formation of NAD+ and glutamate is a measure of the efficiency of ammonia transfer from one active site to the other and of active sites coupling. The stoichiometry data shows that the enzyme achieves maximum efficiency at or below physiologically relevant concentration of L-glutamine and that the synthetase and glutaminase activities are highly synchronized. We have also shown that the observed activation of the glutaminase domain is triggered by the formation of NaAD-AMP intermediate and that binding of the synthetase substrates is not sufficient. We have also purified the C176A variant that lacks any glutamine-dependent activity while it still retains wild-type ammonia-dependent activity. This mutation clearly does not significantly affect catalysis at the synthetase domain. However, the DON modified enzyme retained only 18 % of ammonia-dependent activity. The DON modification might inhibit necessary conformational changes at either domain important for the synthetase activity. An alternative explanation is that DON blocking the ammonia tunnel causes the decreased in activity. The residual activity is due to direct access of ammonia to the synthetase active site. We have crystallized this DON-modified enzyme and we solved the first structure of a glutamine-dependent NAD+ synthetase. The enzyme is a homooctamer of 600 kDa. There are extensive interactions between domains and as a result, one subunit contacts five other subunits in the octamer. We have also identified an intersubunit ammonia tunnel, unique among glutamine amidotransferase enzymes.
