Enzymes in the persistence phase of Mycobacterium tuberculosis

Abstract

As the largest killer of the HIV infected population, and a resident in one third of the global population, research into Mycobacterium tuberculosis (Mtb) is pressing. The pathogenicity of Mtb is attributed to the emergence of drug resistant strains and a quiescent persistent state. The glyoxylate shunt is necessary for the persistence state, and it is not found in humans, making it a promising drug target. In other organisms, entry into this shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). This thesis focuses on mechanistic studies of the enzymes of the glyoxylate shunt, malate synthase (MS) and isocitrate lyase (ICL), and the possible target of regulation, ICDH-1.;MS catalyzes the sequential ordered reaction of glyoxylate and acetyl coenzyme A (AcCoA) to form coenzyme A (CoA) and malate. Primary kinetic isotope effects (KIEs) with [C2H3-methyl]AcCoA ( DV = 1.4 +/- 0.1 and D[V/K[C2H3-methyl]AcCoA ] = 2.3 +/- 0.3) support that the abstraction of the AcCoA proton, and carbon-carbon bond formation to create the intermediate, are rate-limiting. The solvent KIEs (D2OV 1.7 +/- 0.1, D2O[V/K AcCoA] = 1.0 +/- 0.3) likely report on a water generated nucleophile changing the geometry of the malyl-CoA intermediate, in a step following earlier rate-limiting steps.;Isocitrate dehydrogenase catalyzes the reaction of isocitrate and NADP + to CO2, alpha-ketoglutarate (alphaKG), and NADPH. Small primary KIEs and larger solvent KIEs led us to perform the first multiple KIEs on any ICDH, and indicate a stepwise involvement of the two KIEs. In the proposed mechanism, protonation of the enolate by a catalytic acid for formation of alphaKG is rate-limiting. We solved the first x-ray crystal structure of Mtb ICDH-1 to 2.18 A with NADPH and Mn2+ bound. Mtb ICDH-1 was solved by molecular replacement using R132H human cytosolic ICDH. Like this human mutant, Mtb ICDH can also convert alphaKG to alpha-hydroxyglutarate with the concomitant oxidation of NADPH.;ICL catalyses the reversible reaction of isocitrate to glyoxylate and succinate. A large solvent KIE led us to hypothesize that D2O would cause an equilibrium perturbation. We have been able to observe this equilibrium perturbation by D2O directly and continuously with 1H NMR spectroscopy.