Aldose and aldehyde reductase are members of the aldo-keto reductase superfamily, and the former is implicated in the pathogenesis of diabetic cataracts, retinopathy, neuropathy and nephropathy. Aldose reductase inhibitors, proposed for the prevention an therapy of these diabetic complications, are not specific and inhibit these two enzymes as well as well as other members of the superfamily. Our objectives are to compare the atomic structure of both aldose and aldehyde reductase enzymes as well as their catalytic and specific operational mechanisms. We seek a better understanding of the enzymatic mechanism in order to rationally develop highly specific inhibitors unique to each enzyme. We propose to: 1) Determine and compare the atomic structure, cofactor and inhibitor binding, and catalytic mechanisms of human aldose and aldehyde reductase using site-directed mutagenesis, functional evaluation and structure determination by x-ray crystallography; 2) Determine and compare the reaction sequence, rate-limiting steps of the two enzymes by primary isotope effect studies, and the pre-steady state rate constants by stopped-flow measurements; 3) Investigate the function and control of the NADPH holding loop in cofactor exchange and the role of the C-terminal loop in determining substrate specificities; and 4) Characterize athe binding of inhibitors to the active sites of both enzymes, and use the information obtained in these studies to design specific inhibitors of both enzymes, using computer modelling, organic solvent mapping of enzyme surface, peptidomimetic compounds and knowledge of the mechanistic pathways.
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