This proposal addresses the mechanism of action of two flavin- linked enzymes with roles in mitochondrial fatty acid oxidation and in disulfide bond formation in the endoplasmic reticulum. The acyl-CoA dehydrogenases have assumed considerable metabolic importance in genetic deficiencies of fatty acid oxidation and in the bio-activation of cytotoxic fatty acids. The mechanism of chain-length discrimination will be investigated with the medium chain acyl-CoA dehydrogenase, using a variety of acyl-CoA analogues, and employing protein crystallography and kinetic probes of the active site environment. Factors influencing the equilibrium between the """"""""open"""""""" and """"""""closed"""""""" conformations of the enzyme will be studied to understand how the pK of the catalytic base and the reactivity of the flavin prosthetic group are modulated. The mechanism-based inactivation of the medium chain enzyme by a series of cytotoxic 4-thia-acyl-CoA analogues will be examined. The targets of these covalent modification reactions will be determined using protein chemistry and crystallography. The facile inactivation of enoyl-CoA hydratase by several 4-thia- acyl-CoA analogues will be studied using the same methods. The sulfhydryl oxidase from egg white is the best understood member of a newly-recognized protein family with roles in growth regulation in human fibroblasts, in the extracellular matrix, and in spermatogenesis. The oxidase is highly active with a range of reduced protein substrates, and cooperates with protein disulfide isomerase in the generation of the correct disulfide pairings in pancreatic ribonuclease. The mechanism of the reductive half- reaction will be probed with a variety of reduced protein substrates using rapid reaction and static approaches. Flavin analog studies will allow an evaluation of the active site environment in the oxidase, will permit modulation of the internal equilibrium between FAD and redox-active disulfide moieties, and should provide sensitive spectrophotometric tools for the study of complexes between reduced protein substrates and the egg white oxidase.
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