| Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP with the concomitant reduction of NAD+ . This is the rate-limiting and first committed step in de novo guanine nucleotide biosynthesis. IMPDH catalyzes two chemical transformations: a redox reaction involving nucleophilic attack of IMP by Cys319 promoting hydride transfer and forming a covalent E-XMP* intermediate and hydrolysis of E-XMP* producing XMP. The enzymatic base required for water activation is unknown.;Two flexible loops, the loop and flap, contribute to the active site. The role of the flap is not understood but, mutation of a conserved flap Arg abolishes activity in Streptococcus pyogenes IMPDH. The loop contains Cys319 and helps coordinate a K+ ion. Structures of IMPDH exhibit disorder and conformational heterogeneity in the loop and flap, suggesting that conformational change is important to catalysis. The aim of this thesis is to understand the role of these loops and conformational change in the IMPDH reaction.;In this thesis, the role of the flap is elucidated by kinetic characterization of the mutants Arg418Ala and Tyr419Phe. These flap mutations selectively impair hydrolysis and a surprising role for Arg as the general base is suggested. This work also biochemically establishes that the flap serves as a structural switch toggling IMPDH between a dehydrogenase (flap open, NAD+ site exposed) and a hydrolase (flap closed, NAD+ site occluded). Furthermore, differences in the flap equilibrium help account for inhibitor selectivity.;All IMPDHs are activated by K+ though the mechanism of activation is unclear. The loop coordinates K+ and its mechanism of activation is investigated through determination of the K+-dependence of each step in the IMPDH reaction. In contrast to the usual model of allostery, K+ does not affect substrate or product affinity and activation does not occur by acceleration of the chemical transformations. In the absence of K+, the rate-limiting step appears to be a conformational change. K+ accelerates this >65-fold making hydrolysis rate-limiting. This work suggests a novel allosteric mechanism whereby K+ exerts kinetic control over the protein. |
