title: Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase.
abstract: Hydroxymethylglutaryl-CoA synthase-catalyzed condensation of acetyl-CoA with acetoacetyl-CoA requires enolization/carbanion formation from the acetyl C-2 methyl group prior to formation of a new carbon-carbon bond. Acetyldithio-CoA, a readily enolizable analog of acetyl-CoA, was an effective competitive inhibitor of avian hydroxymethylglutaryl-CoA synthase (Ki = 28 microm). In the absence of cosubstrate, enzyme catalyzed the enolization/proton exchange from the C-2 methyl group of acetyldithio-CoA. Mutant enzymes that exhibited impaired formation of the covalent acetyl-S-enzyme reaction intermediate exhibited diminished (D159A and D203A) or undetectable (C129S) rates of enolization of acetyldithio-CoA. The results suggest that covalent thioacetylation of protein, which has not been detected previously for other enzymes that enolize this analog, occurs with hydroxymethylglutaryl-CoA synthase. Enzyme catalyzed the transfer of the thioacetyl group of this analog to 3'-dephospho-CoA suggesting the intermediacy of a covalent thioacetyl-S-enzyme species, which appears to be important for proton abstraction from C-2 of the thioacetyl group. Avian enzyme glutamate 95 is crucial to substrate condensation to form a new carboncarbon bond. Mutations of this invariant residue (avian enzyme E95A and E95Q; Staphylococcus aureus enzyme E79Q) correlated with diminished ability to catalyze enolization of acetyldithio-CoA. Enolization by E95Q was not stimulated in the presence of acetoacetyl-CoA. These observations suggest either a direct (proton abstraction) or indirect (solvent polarization) role for this active site glutamate.
authors.affiliation: Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
authors.forename: Chang-Zeng
authors.initials: CZ
authors.lastname: Wang
authors.forename: Ila
authors.initials: I
authors.lastname: Misra
authors.forename: Henry M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000214:Acyl Coenzyme A / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods*; D001717:Birds; D002244:Carbon / Q000737:chemistry; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D011485:Protein Binding; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology; D013997:Time Factors
chemical_list: D000214:Acyl Coenzyme A; D011522:Protons; D011994:Recombinant Proteins; C059703:acetyldithio-coenzyme A; D018698:Glutamic Acid; D002244:Carbon; D006904:Hydroxymethylglutaryl-CoA Synthase