Diabetes mellitus is a debilitating disease affecting millions of people worldwide, and is the cause of significant morbidity and mortality due to progressive impairment of the visual, renal, nervous and vascular systems. Damage to these tissues results from biochemical and metabolic alterations occurring in response to chronic hyperglycemia. Considerable evidence suggests that products of aldose reductase, the first enzyme of the polyol pathway, may represent a biochemical link between hyperglycemia and diabetic complications in the eye and other major organ systems. Inhibition of aldose reductase provides a therapeutically rational means to delay the-onset or progression of diabetic complications. However, development and use of potent and selective inhibitors will depend on a thorough understanding of the enzyme's catalytic mechanism and the structural features of its active site and inhibitor binding domains. We propose to combine mutagenesis and crystallography studies to evaluate the structural and functional features of human aldose reductase. To evaluate the structural basis for substrate specificity, we will construct and analyze a series of mutants containing structurally conservative replacements of three amino acids at the active site. Kinetic studies comparing the substrate specificity of these mutants with wild type aldose reductase are expected to reveal the structural basis underlying preferential utilization of some substrates. Three-dimensional structures of the enzymes complexed with substrates will also be determined. The binding sites for clinically- relevant aldose reductase inhibitors will also be evaluated by determining inhibition constants using wild type and mutant aldose reductases. Three dimensional structures of aldose reductase complexed with inhibitors will also be determined by x-ray crystallography. The abundance of aldose reductase gene products will be measured in pre- and postpubertal normal and diabetic ovary and testes and compared with other oxidoreductases and steroid metabolizing enzymes to-examine the potential linkage between alterations in aldose reductase gene expression and susceptibility to development of diabetic complications. The structural basis conferring the ability of aldose reductase to utilize steroid substrates will be examined by expression and characterization of chimeric enzymes constructed from aldose reductase and steroid dehydrogenase genes.
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