Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase
Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase
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DOI：
10.1074/jbc.m406566200
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发表时间：
2004-09-24
影响因子：
4.8
通讯作者：
Miziorko, HM
中科院分区：
文献类型：
--
作者：
Wang, CZ;Misra, I;Miziorko, HM
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 (K-i = 28 muM). 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 carbon-carbon 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.
