The determination of the relationship between an enzymes' three- dimensional structure and its catalytic function is the long-range objective of this research. We have selected, as appropriate system for such investigations, a number of enzymes in the flavoprotein reductase family, which contain some of the most mechanistically intriguing and physiologically important enzymes found in nature. They serve key roles in oxidative stress management in humans and bacteria (erythrocyte glutathione reductase and macrophage alkyl hydroperoxide reductase, NADH peroxidase), and in trypanosomes (trypanothione reductase). They serve to detoxify heavy metals (mercuric reductase) and in mammalian liver to provide substrates for xenobiotic removal (glutathione reductase). They play key roles in carbohydrate metabolism (lipoamide dehydrogenase) and have been suggested to participate in the proper folding of proteins via catalysis of disulfide bond formation and rearrangement (thioredoxin reductase and the E. coli dsbA gene product). The amino acid sequences of all of these proteins have been determined, and the high resolution three dimensional structures of six members of the family, including all the enzymes under investigation, have been reported. Evidence has been obtained supporting a common rate-limiting proton transfer occurring in of the oxidative half-reactions for four of these enzymes. The transition state for hydride transfer between reduced pyridine nucleotide and flavin has been determined for glutathione reductase, and a mechanism for enzymatic transition state stabilization has been proposed based on our studies and the structure of the enzyme-nucleotide binary complex. the extension of these studies with human erythrocyte glutathione reductase and trypanothione reductase from the parasitic protozoan, Trypanosoma congolense, will include the testing of our hypothesis using a combination of kinetic, isotopic, and mutagenic approaches. In both these systems, the involvement of an ion pair between a glutamate carboxyl and a lysine amino group in hydride ion stabilization will be assessed by mutagenesis of the glutamate to a glutamine or aspartate residue. Structural and isotopic investigations of the most mechanistically unique, and enigmatic, of the reductases, NADH peroxidase, will be continued. The enzymes discussed represent ideal subjects for the detailed structure/function investigations proposed in this grant due to the availability of cloned, sequenced and overexpressed genes, high resolution three-dimensional structures, and favorable spectroscopic and isotopic properties. These studies will provide a detailed description of the molecular forces that are utilized by these enzymes to stabilize the transition state for hydride transfer, one of the simplest, but most central, chemical redox reactions in biology.
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