This program's long term objective is an understanding, at the molecular level, of the mechanisms which control metabolic flux through ketogenesis and the related early steps in cholesterogenesis. Disturbances in the ketogenic pathway can lead to developmental abnormalities as well as ketoacidosis; two inborn errors of metabolism, that have serious medical consequences, mapped within the ketogenic pathway. Likewise, impaired control of cholesterogenesis can produce hypercholesterolemia and the array of problems derived from vascular deposition of excess cholesterol. Enzymes under investigation include hydroxymethylglutaryl-CoA (HMG-CoA) synthase and HMG-CoA lyase will be investigated to elucidate structure/function correlations that account for the catalytic activity and regulatory properties. For each of these, the investigators have developed recombinant proteins and initiated extensive steps in characterizing these proteins. For HMG-CoA synthase, site directed mutagenesis has implicated several active site amino aids in catalysis. Several aims address functional assignments for these and any other residues identified as important to the reaction chemistry. The assignments of roles for these residues will be pursued using mechanistic and physical biochemistry approaches. Expression and functional assignment of prokaryotic proteins that are homologous to animal HMG-CoA synthase will also be explored. Any suitable candidates will be screened in crystallization trials aimed at securing the first diffraction quality crystals for this protein. In the case of HMG-CoA lyase, directed mutagenesis work has implicated amino acids involved in regulation and catalysis.
Specific aims to be pursued include extension of this approach to new targets implicated by protein chemistry, sequence homology, or mapping of point mutations that result in human HMG-CoA lyase deficiency. The mutants which appear to be a mechanistically informative will be characterized using mechanistic and physical approaches similar to those previously employed in this laboratory to identify catalytic or regulatory residues. The significance of thiol/disulfide exchange, in vitro regulation of peroxisomal HMG-CoA lyase, and in vivo regulation of mitochondrial HMG-CoA lyase will be tested. Crystalization trials will be extended in an attempt to secure diffraction quality crystals of prokaryotic or eukaryotic HMG-CoA lyase.
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