Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage.
Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.
Kim
Y T
YT
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Churchich
J E
JE
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D013439:Sulfhydryl Reagents; D013552:Swine; D014357:Trypsin; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives
D046911:Macromolecular Substances; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; C038560:4-dimethylaminoazobenzene-4'-iodoacetamide; D004124:p-Dimethylaminoazobenzene; D000612:4-Aminobutyrate Transaminase; D014357:Trypsin; D003545:Cysteine