Expression, characterization and structure/function analysis of avian cytosolic 3-hydroxy-3-methylglutaryl coenzyme A synthase

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA to produce HMG-CoA, a key intermediate in ketogenesis and cholesterogenesis. As anticipated for the enzyme that catalyzes the first irreversible step in these metabolic pathways, HMG-CoA synthase has been implicated as a control point. The cholesterogenic isozyme, located in the cytosol, has recently become a subject of increased interest as a target for pharmacological intervention to modulate serum cholesterol levels. Despite the significance of the HMG-CoA synthase reaction, many details concerning the reaction chemistry remain to be elucidated. The main objective of this project was to further our understanding of the mechanism of the HMG-CoA synthase reaction. Site-directed mutagenesis was employed to allow assignment of function(s) to the active site amino acids. In order to engineer site-specific mutations and to produce avian cytosolic synthase and its variants in adequate amounts for structural and mechanistic studies, a recombinant expression system utilizing a bacteriophage T7 promoter was designed. The target protein was expressed in E. coli in a soluble form representing over 20% of the total cellular protein. Overexpression has facilitated the isolation of HMG-CoA synthase in homogenous form. The properties of the recombinant enzyme were found to be equivalent to the liver cytosolic enzyme, suggesting its utility as a model for structural and mechanistic studies. These observations also verify our prediction that the cDNA encodes the cholesterogenic isozyme.; Site-specific substitution was performed to generate a C129S variant in which the cysteine previously implicated in formation of an acetyl-S-enzyme intermediate was replaced by a serine. C129S synthase exhibits a 10{dollar}sp5{dollar}-fold diminution in k{dollar}sb{lcub}rm cat{rcub}{dollar} while retaining its ability to form noncovalent complexes with acetyl-CoA and a spin labeled acetyl-CoA analog. The diminution in k{dollar}sb{lcub}rm cat{rcub}{dollar} exhibited by C129S synthase is attributable to its inability to form a covalent acetyl-enzyme intermediate, supporting our previous assignment of this function to C129. The data also demonstrate a stringent requirement of the enzyme for a sulfhydryl functionality in the formation of an acetyl-enzyme intermediate.; Previous chemical modification of C129S synthase with a mechanism-based inhibitor has revealed the presence of another reactive cysteine in the active site. Furthermore, cysteine has been implicated in the acid/base catalysis of some enzymatic reactions. To evaluate the role of additional cysteines in HMG-CoA synthase reaction, each of the invariant cysteines was replaced by an alanine. Kinetic characterization of the isolated mutants indicated that none of the additional cysteines is essential for HMG-CoA synthesis.; A model reaction that appeared useful for comparison with the synthase reaction is the well characterized citrate synthase-catalyzed condensation of acetyl-CoA and oxaloacetate. A tentative assignment of two active site histidines and one aspartate has been made in this enzyme. To test the role of histidines in HMG-CoA synthase reaction, each of the 3 conserved histidines in HMG-CoA synthase was replaced by an asparagine. H264N mutant exhibits some interesting differences from the wild type enzyme. The V{dollar}sb{lcub}rm max{rcub}{dollar} for H264N is decreased by 40-fold and the stoichiometry of acylation is decreased by a factor of 10, whereas the binding of the spin labeled acetyl-CoA analog is indistiguishable from the wild type enzyme. These observations may be ascribed to decreased stabilization of the acetyl-S-enzyme intermediate.