This proposal is to extend the structural studies on the medium chain acyl- CoA dehydrogenase (MCAD) of pig liver to obtain a more detailed picture of this enzyme and to determine structures of related enzymes, the human MCAD, the porcine short chain acyl CoA dehydrogenase (SCAD) and a similar enzyme from ma bacterium. These are members of a family of enzymes that carry out the first oxidative step in the catabolism of fatty acids, the principal fuel for many organs, including liver, kidney, heart and skeletal muscle. The rate o oxidation can be altered by diet, physiological state and disease, exemplified by starvation, pregnancy and diabetes. The critical role of these enzymes in metabolism is illustrated by the severity of human diseases attributed to inherited deficiencies of each of the three dehydrogenases. The MCAD from pig liver mitochondria has recently been studies intensively and progress has been made in elucidating the catalytic, structural and cellular properties of the enzyme. Detailed structural information from high resolution X-ray analysis will enable us to relate chemical functions to the structure of the protein and to define the catalytic mechanism. The enzyme has a molecular weight of 172,000 and contains four identical subunits, each containing one equivalent of flavin adenine dinucleotide (FAD). Three-dimensional X-ray analysis of the enzyme at 3A resolution has revealed the location and orientation of the FAD and the conformation of the entire peptide chain. The polypeptide folding near the FAD binding site is different from the structures seen in other flavoproteins. It is proposed to extend the structural analysis to 2A resolution and to elucidate the detailed mechanism of oxidation of acyl-CoA thioesters and transfer of electrons to the electron transfer flavoprotein, the physiological oxidant of he dehydrogenase in mitochondria. It is also proposed to initiate both enzymatic and crystallographic studies on the human MCAD; this enzyme has been cloned and the studies will employ both the native protein and altered proteins made by site-directed mutagenesis. Crystallographic studies will also be carried out on the SCAD of pig liver and on the butyryl-CoA dehydrogenase of M. elsdenii, which has already been crystallized in a form suitable for X-ray analysis. Comparisons of these structures will enable us to ascertain the structures involved in the general catalytic mechanisms of this group of enzymes and the features that determine the specificity of each acyl-CoA dehydrogenase.
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