Inhibition of Inosine-5′-monophosphate Dehydrogenase from Bacillus anthracis: Mechanism Revealed by Pre-Steady-State Kinetics

Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion Of inosine 5'-monophosphate (IMP) to xanthosine S'-monophosphate (XMP). The enzyme is, an emerging target for antimicrobial therapy. The. small molecule inhibitor A110 has. been identified as a potent and selective inhibitor of IMPDHs from a variety of pathogenic microorganisms. A recent X-ray crystallographic study reported that the inhibitor binds to the NAD+ cofactor site and forms a ternary complex with IMP. Here we report a pre-steady-state stopped-flow-kinetic investigation of IMPDH from Bacillus anthracis designed to assess the kinetic significance of the crystallographic results. Stopped-flow kinetic experiments defined nine microscopic rate constants and two equilibrium,constants that characterize both the catalytic cycle and, details of the inhibition mechanism. In combination with steady-state initial rate studies, the results show that the inhibitor binds with high affinity (K-d approximate to 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (K-d approximate to 1 mu M) is observed,between the-inhibitor and E. IMP. Thus, the E.IMP.A110 ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant.