Identification of the site of acetyl-S-enzyme formation on avian liver mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

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Abstract
Avian liver mitochondrial hydroxymethylglutaryl-CoA synthase contains an active-site cysteine involved in forming the labile acetyl-S-enzyme intermediate. Identification of and assignment of function to this cysteine have been accomplished by use of an experimental strategy that relies upon generation and rapid purification of the S-acetylcysteine-containing active-site peptide under mildly acidic conditions that stabilize the thioester adduct. Automated Edman degradation techniques indicate the peptide''s sequence to be Arg-Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-Thr-Asn-Ala-Cys-Tyr. The acetylated cysteine corresponds to position 129 in the sequence deduced from cDNA data for the hamster cytosolic enzyme [Gil, G., Goldstein, J. L., Slaughter, C. A., and Brown, M.S. (1986) J. Biol. Chem. 261, 3710-3716]. The acetyl-peptide sequence overlaps that reported for a tryptic peptide that contains a cysteine targeted by the affinity label 3-chloropropionyl-CoA [Miziorko, H. M., and Behnke, C. E. (1985) J. Biol. Chem. 260, 13513-13516]. Thus, availability of these structural data allows unambigous assignment of the acetylation site on the protein as well as a refinement of the mechanism explaining the previously observed affinity labeling of the enzyme.

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