The pyruvate-dehydrogenase complex from Azotobacter vinelandii.
The pyruvate dehydrogenase complex from Axotobacter vinelandii was isolated in a five-step procedure. The minimum molecular weight of the pure complex is 600,000, as based on an FAD content of 1.6 nmol-mg protein-1. The molecular weight is 1.0-1.2 X 10(6), indicating 1 mole of lipoamide dehydrogenase dimer per complex molecule. Sodium dodecylsulphate gel electrophoretical patterns show that apart from pyruvate dehydrogenase (Mr89,000) and lipoamide dehydrogenase (Mrmonomer 56,000) two active transacetylase isoenzymes are present with molecular weight on the gel 82,000 and 59,000 but probably actually lower. The pure complex has a specific activity of the pyruvate-NAD+ reductase (overall) reaction of 10 units-mg protein-1 at 25 degrees C. The partial reactions have the following specific activities in units-mg protein-1 at 25 degrees C under standard conditions: pyruvate-K3Fe(CN)6 reductase 0.14, transacetylase 3.6 and lipoamide dehydrogenase 2.9. The properties of this complex are compared with those from other sources. NADPH reduced the FAD of lipoamide dehydrogenase as well in the complex as in the free form. NADP+ cannot be used as electron acceptor. Under aerobic conditios pyruvate oxidase reaction, dependent on Mg2+ and thiamine pyrophosphate, converts pyruvate into CO2 and acetate; V is 0.2 mumol 02-min-1-mg-1, Km(pyruvate)0.3 mM. The kinetics of this reaction shows a linear 1/velocity-1/[pyruvate] plot. K3Fe(CN)6 competes with the oxidase reaction. The oxidase activity is stimulated by AMP and sulphate and is inhibited by acetyl-CoA. The partially purified enzyme contains considerable phosphotransacetylase activity. The pure complex does not contain this activity. The physiological significance of this activity is discussed.
D000123:Acetyltransferases / Q000302:isolation & purification / Q000378:metabolism; D000332:Aerobiosis; D001395:Azotobacter / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D010101:Oxygen Consumption; D011768:Pyruvate Dehydrogenase Complex / Q000302:isolation & purification / Q000378:metabolism*; D011771:Pyruvate Oxidase / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D011768:Pyruvate Dehydrogenase Complex; D005182:Flavin-Adenine Dinucleotide; D011771:Pyruvate Oxidase; D009247:NADH, NADPH Oxidoreductases; D008058:Dihydrolipoamide Dehydrogenase; D000123:Acetyltransferases; D008274:Magnesium