The reversible synthesis of acetoacetyl-CoA from two molecules of acetyl-CoA is catalyzed by a set of three mammalian thiolase isozymes. The three isozymes are involved in different metabolic pathways central to the metabolism of acetyl-CoA, including isoprenoid and steroid biosynthesis, fatty acid beta oxidation, and ketone body synthesis and degradation. The long-term goal of this research is to delineate the chemical and structural factors contributing to efficient catalysis of the proton and acyl transfer reactions of these enzymes. The mechanism of these reactions is of considerable practical utility in the design of specific mechanism based inhibitors for thiolase and other acyl-CoA utilizing enzymes. Recent studies have revealed a complex and unusual arrangement of at least two and likely three proximal sulfhydryl groups at or near the active site. We propose to explore the mechanistic and potential regulatory significance of this unusual sulfhydryl cluster. The identity of the various sulfhydryl groups involved in enzyme oxidation and other specific reactions such as an abortive acylation by hydroxybutyryl-CoA will be determined by developing a peptide mapping technique to visualize only sulfhydryl containing peptides. The potential involvement of mutiple acyl transfers between sulfhydryl groups in the normal reaction catalyzed by by thiolase will be investigated by the application of rapid quench experiments. The involvement of a thiolimidate intermediate in catalysis of acetyl-CoA enolization will be probed by analyzing the exchange (or lack of) of the carbonyl oxygen of acetyl-CoA with 180 water using the isotope effect on the 13C NMR chemical shift of the C-1 of acetyl-CoA. Differences in sulfhydryl chemistry including thiol/disulfide redox potentials and chemical reactivity between the three mammalian thiolases will be examined.
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