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

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 ( Mt IMPDH) catalyzes the oxidation of IMP to XMP, with concomitant conversion of NAD + to NADH. In the present work, the guaB2 -encoded Mt IMPDH has been cloned, expressed and purified to homogeneity. The recombinant Mt IMPDH 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 Mt IMPDH predominates as a tetramer. Steady-state kinetics showed that Mt IMPDH 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 p K 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.