Utility of acetyldithio-CoA in detecting the influence of active site residues on substrate enolization by 3-hydroxyl-3-methylglutaryl-CoA synthase.

The Journal of Biological Chemistry
Chang-Zeng WangHenry M Miziorko

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....Continue Reading

References

Apr 1, 1991·Biochemical Medicine and Metabolic Biology·L V WrensfordV E Anderson
Feb 21, 1989·Biochemistry·I D Wlassics, V E Anderson
Jun 1, 1967·European Journal of Biochemistry·H Eggerer, A Klette

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Citations

Oct 12, 2010·Archives of Biochemistry and Biophysics·Henry M Miziorko

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