Mapping of reactive sulfhydryls in avian liver 3-hydroxy-3-methylglutaryl coenzyme A synthase.
Avian liver mitochondrial 3-hydroxy-3-methylglutaryl coenzyme A synthase contains seven sulfhydryls per 53 kDa subunit. Peptides that harbor these sulfhydryls can be mapped by reverse-phase HPLC separation of tryptic digests of denatured 14C-carboxymethylated enzyme. Native enzyme is inactivated by a variety of reagents that target cysteine residues. Of particular interest is the enzyme's sensitivity to reagents (e.g., CdCl2, copper phenanthroline) that target vicinal thiols. The identity of the cysteines which are modified by these reagents can be determined by peptide mapping after denaturation. 14C-carboxymethylation and trypsin digestion of the sample. While the extent of reaction of any particular cysteinyl sulfhydryl depends on the identity of the reagent employed, three of the protein's seven cysteinyl sulfhydryls are frequently modified upon inactivation of the enzyme. The peptides which contain these reactive sulfhydryls have been isolated and their sequences have been determined by Edman degradation techniques. Comparison of these sequences with the deduced primary structure of the rodent cytosolic enzyme (Gil et al. (1986) J. Biol. Chem. 261, 3710) indicates strong homologies. These homologies allow assignment of the reactive residues as Cys-129, Cys-224 and Cys-268. The sensitivity of these residues to reagents that target vicinal thiols, coupled with the fact that cys-129 is the residue involved in formation of the acyl-S-enzyme intermediate (Vollmer et al. (1988) Biochemistry 27, 4288), suggests that these three residues may be closely juxtaposed within the enzyme's catalytic domain.
Miziorko
H M
HM
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Behnke
C E
CE
Wang
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds; D002851:Chromatography, High Pressure Liquid; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D010455:Peptides; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D006904:Hydroxymethylglutaryl-CoA Synthase