AMINO-ACID SEQUENCE OF AN ACTIVE-SITE PEPTIDE OF AVIAN LIVER MITOCHONDRIAL 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE

  • 1 January 1985
    • journal article
    • research article
    • Vol. 260  (25) , 3513-3516
Abstract
Hydroxymethylglutaryl-CoA synthase is irreversibly inhibited by the active site-directed inhibitor 3-chloropropionyl-CoA. Enzyme modification has been postulated to involve alkylation of an active site cysteinyl sulfhydryl group. DEAE-Sephadex chromatography of tryptic digests prepared from enzyme inactivated using chloro[14C]propionyl-CoA suggested that bound radioactivity is localized on one peptide. Specificity of the modification was further demonstrated by reverse-phase high pressure liquid chromatography, which was used to isolate the radioactively labeled peptide in a chemically homogeneous form. Automated gas-phase Edman degradation techniques have been employed to confirm the assignment of cysteine as the inhibitor''s target residue and to elucidate the sequence of amino acids which flank the 14C-carboxyethylated cysteine: Glu-Ser-Gly-Asn-Thr-Asp-Val-Glu-Gly-Ile-Asp-Thr-(Thr)-Asn-Ala-S-[14C]carboxyethylcysteine-Try-Gly-Gln-Thr-(Ala). These data represent the first assignment of active site structure for hydroxymethylglutaryl-CoA synthase.