This program's long term objective is an understanding, at a molecular level, of the mechanisms which control ketogenesis and the related early steps in cholesterogenesis. Disturbances in the ketogenic pathway can lead to developmental abnormalities as well as ketoacidosis; an inborn error of metabolism that can have serious medical consequences has been mapped within this pathway. Likewise, impaired control of the cholesterogenic pathway can produce hypercholesterolemia and the array of problems derived from vascular deposition of excess cholesterol. Several enzymes are currently under investigation. The structural information available for these enzymes varies widely. For each enzyme, plans are outlined to secure additional structural information and to eventually define structure/function correlations using protein modification and/or recombinant DNA methodology. For hydroxymethylglutaryl-CoA (HMG-CoA) synthase, the primary structure and some elements of active site structure have been elucidated. While additional structural information makes it now practical to develop capabilities for producing a recombinant enzyme. Such an accomplishment would allow protein engineering work aimed at testing structure/function hypotheses. In the case of HMG-CoA lyase, available structural information is currently limited, but there are excellent prospects for a substantial expansion of this data base in the near future. As these data become available, this protein will also be targeted for investigation of structure/function relationships. Point mutations in HMG-CoA lyase are likely to account for many of the clinically documented defects in HMG-CoA metabolism. As structural consequences of genetic mutations are defined, our understanding of lyase function will be enhanced by enzymological characterization of both the defective proteins and of engineered variants of the naturally occurring mutants. Protein chemistry work will provide additional insight into the mechanisms of action and of regulation. Finally, the enzyme that activates ketone bodies for use in biosynthesis, acetoacetyl-CoA synthetase, is poorly characterized. A reliable, efficient purification procedure will be developed. Enzyme will be thoroughly characterized, expediting investigation of its primary and active site structure by protein chemistry and molecular biology techniques.
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