title: Inhibition of Inosine-5'-monophosphate Dehydrogenase from Bacillus anthracis: Mechanism Revealed by Pre-Steady-State Kinetics.
abstract: 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 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 (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.
authors.affiliation: Department of Biology, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Wei
authors.affiliation: Department of Biology, Brandeis University , Waltham, Massachusetts 02454, United States.|BioKin Ltd. , Watertown, Massachusetts 02472, United States.
authors.forename: Petr
authors.initials: P
authors.lastname: Kuzmič
authors.affiliation: Department of Chemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Runhan
authors.initials: R
authors.lastname: Yu
authors.affiliation: Department of Biology, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Gyan
authors.initials: G
authors.lastname: Modi
authors.affiliation: Department of Biology, Brandeis University , Waltham, Massachusetts 02454, United States.|Department of Chemistry, Brandeis University , Waltham, Massachusetts 02454, United States.
authors.forename: Lizbeth
authors.initials: L
authors.lastname: Hedstrom
mesh_terms: D001408:Bacillus anthracis / Q000201:enzymology*; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008958:Models, Molecular
chemical_list: D007168:IMP Dehydrogenase