Utility of Acetyldithio-CoA in Detecting the Influence of Active Site Residues on Substrate Enolization by 3-Hydroxyl-3-methylglutaryl-CoA Synthase
| Autor: | Ila Misra, Chang-Zeng Wang, Henry M. Miziorko |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Hydroxymethylglutaryl-CoA Synthase
Staphylococcus aureus Time Factors Stereochemistry Glutamic Acid Reaction intermediate Biochemistry Catalysis Cofactor Birds chemistry.chemical_compound Animals Molecular Biology Carbanion chemistry.chemical_classification Binding Sites Dose-Response Relationship Drug biology ATP synthase Active site Cell Biology Hydrogen-Ion Concentration Carbon Recombinant Proteins Kinetics Enzyme Models Chemical chemistry Covalent bond Mutation biology.protein Acyl Coenzyme A Protons Protein Binding Methyl group |
| Zdroj: | Journal of Biological Chemistry. 279:40283-40288 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m406566200 |
| Popis: | 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 μm). 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. |
| Databáze: | OpenAIRE |
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