The objective is an understanding, at a molecular level, of the mechanisms which control ketogenesis and the related early steps in cholesterogenesis. Disturbances in control of the former pathway can lead to developmental and neurological abnormalities as well as ketoacidosis. Impaired control of the latter pathway can produce hypercholesteremia and the variety of problems related to vascular deposition of excess cholesterol. The hydroxymethylglutaryl-CoA (HMG-CoA) cycle accounts for ketone body production in liver. The enzymes that catalyze the two irreversible steps in this cycle, HMG-CoA synthase and HMG-CoA lyase, are available in homogeneous form in this laboratory, facilitating investigation of their interaction and of the chemical events involved in ketogenesis. Critical active site amino acids are being identified by development and application of affinity labels and mechanism based inhibitors. Specific modifications will lead not only to identification of the target residues, but also to elucidation of active site structure by generating active site peptides containing the target residues and determining the amino acid sequence of the peptides. The positions of key amino acides within the modified proteins will be mapped using an approach that relies on end labeling and partial specific cleavages. Orientation of active site residues suspected of being in close proximity in the native enzyme will be investigated using crosslinking techniques. Organizatin of ketogenic and cholesterogenic enzymes may account for the efficient flux of metabolites through these pathways. Channeling of aceto-acetyl-CoA between the enzymes which synthesize and utilize this metabolite will be evaluated for ketogenic and cholesterogenic pathways using kinetic approaches in studies on the appropriate cell fractions.
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