Yang Wei, Petr Kuzmič, Runhan Yu, Gyan Modi, Lizbeth Hedstrom

Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the conversion of inosine-5'-monophosphate (IMP) to xanthosine- 5'-monophosphate (XMP). The enzyme is an emerging target for antimicrobial therapy. The small molecule inhibitor A110 has been identified as 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 (Kd ~ 50 nM) predominantly to the covalent intermediate on the reaction pathway. Only a weak binding interaction (Kd ~ 1 µM) is observed between the inhibitor and E.IMP. Thus the E.IMP.A110 ternary complex, observed by X-ray crystallography, is largely kinetically irrelevant.