The overall aim of this project is to understand the structural basis for efficient enzymic catalysis of hydrogen transfer from weakly acidic carbon and oxygen centers. We have selected several representative systems for study. For proton transfer catalysis: phosphoglucose isomerase (PGI), which first catalyzes proton transfer to oxygen (ring-opening) and then uses acid/base catalyzed proton transfer between carbons to isomerize a sugar phosphate, and D-galactose mutarotase (GalM), which catalyses ring-opening and proton transfer between oxygens on a sugar substrate that is not phosphorylated. For hydride transfer: xylose isomerase (XyI), which transfers hydrogen between carbon centers via metal-mediated 1,2-hydride shift and also catalyzes proton transfer between oxygens, i.e., sugar ring opening; and inosine 5'-monophosphate dehydrogenase (IMPDH), which uses NAD+ as a hydride acceptor in the transformation of inosine to xanthosine. To test the putative roles of various residues in the mechanisms of these enzymes, we will use a combination of site-directed mutagenesis and X-ray crystallography. In addition to an understanding of the precise chemical and structural roles of the residues in the active sites, these studies will provide views of the reaction pathways and a qualitative and semiquantitative assessment of the contribution to proton and hydride transfer catalysis of factors such as: general-acid/general-base catalysis; electrophilic catalysis; short, strong hydrogen bonds to the transition state; electrostatic stabilization of charged species, participation of bound water molecules, and substrate/cofactor strain. Understanding the mechanisms of action of these enzymes also has important consequences for human health. PGI is mutated in a severe hereditary metabolic disease. In addition, PGI by some unknown mechanism is secreted from the cell where it moonlights as a potent cytokine and tumor cell meidator. XyI is important in food production and is also being used in biotechnology. Ga1M is part of the Leloir pathway for the utilization of galactose and there are many known galactose metabolic disorders. IMPDH catalyzes the rate-determining step in GMT biosynthesis and is a target for immunosuppressive, anticancer, antiviral and antimicrobial drugs.
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