Biochemical characterization of Mycobacterium tuberculosis IMP dehydrogenase: kinetic mechanism, metal activation and evidence of a cooperative system

Abstract

Enzymes from the nucleotide biosynthesis pathway are potential targets for the development of novel anti-mycobacterial agents. Inosine 5′-monophosphate (IMP) dehydrogenase from Mycobacterium tuberculosis (MtIMPDH) catalyzes the oxidation of IMP to XMP, with concomitant conversion of NAD⁺ to NADH. In the present work, the guaB2-encoded MtIMPDH has been cloned, expressed and purified to homogeneity. The recombinant MtIMPDH has a subunit molecular mass of 54 775 Da, and Inductively Coupled Plasma Optical Emission Spectroscopy and Flame Atomic Absorption Spectroscopy identified a K⁺ ion per subunit. Glutaraldehyde cross-linking data suggest that MtIMPDH predominates as a tetramer. Steady-state kinetics showed that MtIMPDH optimal activity is dependent on the presence of a monovalent cation, mainly K⁺. Initial velocity and product inhibition patterns suggest a steady-state ordered Bi Bi kinetic mechanism in which IMP binds first followed by NAD⁺, and product release is ordered. Hydride transfer appears not to be rate-limiting. The pH-rate profile indicates one deprotonated group essential for catalysis and that groups with pK values of 7.5 and 9.0 are important for NAD⁺ binding. Temperature studies were employed to determine the activation energy of the reaction. The data presented here are discussed in light of the kinetic and structural information available for IMPDHs.