An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart.
1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001322:Autoanalysis; D001345:Autoradiography; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003545:Cysteine / Q000378:metabolism; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis*; D004590:Electrophoresis, Paper; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007461:Iodoacetates; D007655:Ketoglutarate Dehydrogenase Complex / Q000302:isolation & purification; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D012967:Sodium Dodecyl Sulfate; D013438:Sulfhydryl Compounds; D013552:Swine
D000596:Amino Acids; D002250:Carbon Radioisotopes; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D012967:Sodium Dodecyl Sulfate; D007655:Ketoglutarate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; D003545:Cysteine