Purification and properties of the pyruvate dehydrogenase complex from Salmonella typhimurium and formation of hybrids with the enzyme complex from Escherichia coli.
The pyruvate dehydrogenase (Pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1) complex from Salmonella typhimurium was purified, characterized and compared to the enzyme complex from Escherichia coli. No difference could be found in the molecular weights of the native enzyme complexes or in the single polypeptide chains of the enzymes of the two organisms. Values of 100 000, 87 000 and 56 000 were obtained for the polypeptide chains of the pyruvate dehydrogenase, the dihydrolipoamide transacetylase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) and the dihydrolipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) components, respectively. Complete cross-reactivity was found with antibodies directed against the pyruvate dehydrogenase complex from E. coli and electron micrographs of both enzyme complexes reveal identical structures. A high Michaelis constant for pyruvate with a Km = 6 . 10(-4) M and a somewhat weaker cooperativity as compared to the enzyme from E. coli reflect some minor differences, while the binding of the cofactor thiamine diphosphate (Km = 1 . 10(-6) M) is identical for both enzyme complexes. Reassociation to a fully active complex molecule works with equal facility between the pyruvate dehydrogenase component and a dihydrolipoamide transacetylase: dihydrolipoamide dehydrogenase subcomplex from either organism in all possible combinations.
D000123:Acetyltransferases / Q000302:isolation & purification; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification; D051047:Dihydrolipoyllysine-Residue Acetyltransferase; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008854:Microscopy, Electron; D008970:Molecular Weight; D055503:Protein Multimerization; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013045:Species Specificity
D011768:Pyruvate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase; D000123:Acetyltransferases; D051047:Dihydrolipoyllysine-Residue Acetyltransferase