Dr. David R. J. Palmer2010-06-022013-01-042012-06-182013-01-042010-062010-06-17June 2010http://hdl.handle.net/10388/etd-06022010-111229Inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis catalyzes the reversible NAD+-dependent oxidation of the axial hydroxyl group of myo-inositol to form 2-keto-myo-inositol, NADH and H+. IDH is the first enzyme in catabolism of myo-inositol, and Bacillus subtilis is able to grow on myo-inositol as the sole carbon source. Our laboratory has previously shown that this enzyme has an unusual active site that can accommodate large hydrophobic substituents at 1L-4-position of myo-inositol. In this dissertation, the further characterization of this IDH is described, with focus on the mechanism, inhibition, kinetics, substrate binding, and alteration of substrate specificity. A kinetic isotope effect study revealed that the chemical step of the reaction was not rate-limiting. In order to probe the inositol-binding site, five inositol analogues were synthesized and evaluated as competitive inhibitors. Recently the crystal structures of the apo-IDH, holo-IDH and ternary complex have been solved. Using structural information, as well as modeling and sequence alignment approaches, we predicted the active site structure of the enzyme. On the basis of these predictions, coenzyme specificity was converted from entirely NAD+-dependent to 6-fold preference for NADP+ over NAD+ by site-directed mutagenesis. The critical residues for coenzyme recognition were therefore identified. Besides coenzyme specificity alteration, eleven amino acid residues in and around the proposed myo-inositol active site were also modified to test their roles in order to improve our understanding of substrate binding and activation.en-USkineticsinositol dehydrogenasemechanisminhibitionmutagenesistext