3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE - PARTICIPATION OF ACETYL-S-ENZYME AND "ENZYME-S-HYDROXYMETHYLGLUTARYL-SCOA INTERMEDIATES IN REACTION

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  • 1 January 1977
    • journal article
    • research article
    • Vol. 252  (4) , 1414-1420
Abstract
Acetyl-CoA tests stoichiometrically with a cysteinyl sulfhydryl group of avian liver 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase to yield acetyl-S-enzyme. Evidence that acetyl-S-enzyme condenses with the 2nd substrate, acetoacetyl-CoA, to form enzyme-S-HMG-SCoA was obtained by trapping and characterizing this putative intermediate. [14C]Acetyl-S-enzyme was incubated briefly at -25.degree. with acetoacetyl-CoA, precipitated with trichloroacetic acid, and the labeled acylated enzyme species were isolated. Performic acid oxidation of the precipitated [14C]acetyl-S-enzyme intermediates produced volatile [14C]acetic acid from unreacted [14C]acetyl-S-enzyme and nonvolatile [14C]3-hydroxy-3-methyl glutaric acid from enzyme-S-[14C]HMG-SCoA. Condensation of unlabeled acetyl-S-enzyme with [14C]acetoacetyl-CoA or acetoacetyl-[3H]CoA also produced labeled enzyme-S-HMG-SCoA. Thus, the acetyl moiety from acetyl-CoA and the acetoacetyl and CoA moieties from acetoacetyl-CoA all are incorporated into the HMG-CoA which is covalently-linked to the enzyme. Enzyme-S-[14C]HMG-SCoA was subjected to proteolytic digestion under conditions favorable for intramolecular S to N acyl transfer in the predicted cysteine-S-[14C]HMG-SCoA fragment. Performic acid oxidation of the protease-digested material yields N-[14C]HMG-cysteic acid indicating that HMG-CoA was covalently bound to the enzyme via the -SH of an active site cysteine. An isotope trapping technique was employed to test the kinetic competence of acetyl-S-enzyme as an intermediate in the HMG-CoA synthase-catalyzed reaction. Evidence is presented which indicates that the rate of condensation of acetoacetyl-CoA with acetyl-S-enzyme to form enzyme-S-HMG-SCoA is more rapid than either the acetylation of the synthase by acetyl-CoA or the overall forward reaction leading to HMG-CoA. These observations, together with indirect evidence that hydrolysis of enzyme-S-HMG-SCoA is extremely rapid, suggest that acetylation of synthase is the rate-limiting step in HMG-CoA synthesis.