Pyruvate dehydrogenase complex of Escherichia coli. Thiamin pyrophosphate and NADH-dependent hydrolysis of acetyl-CoA.
When the pyruvate dehydrogenase complex of Escherichia coli is reduced by NADH and alkylated by N-[14C]ethylmaleimide, 19-20 nmol of N-[14C]ethylmaleimide are bound per mg of complex. This is in accord with the presence of 10 nmol of functional lipoyl moieties per mg of complex as previously reported. Thus the lipoyl groups are all coupled via dihydrolipoyl dehydrogenase (E3) to reduction by NADH. As previously reported, the complex reductively acetylated by pyruvate and containing 10 nmol of acetyldihydrolipoyl groups per mg of complex produces about 5 nmol of NADH/mg of complex when challenged with CoA and NAD+ in a fast burst. Under anaerobic conditions a slow secondary process extending over 1 h produces another 5 nmol of NADH/mg of complex. The relationship between the two classes of acetyldihydrolipoyl groups is unknown but could reflect either intrinsic structural inequivalence of lipoyl groups (2/subunit of dihydrolipoyl transacetylase, E2). Alternatively, the acetyldihydrolipoyl groups may undergo reversible isomerization to structurally distinct forms. The purified complex catalyzes the cleavage of acetyl-CoA by two processes. The trace contaminant phosphotransacetylase catalyzes cleavage by phosphate to acetyl-P. The complex itself catalyzes hydrolysis of acetyl-CoA in a reaction that requires all three enzymes, NADH, thiamin pyrophosphate, and the lipoyl groups of E2. The hydrolytic pathway evidently involves overall reversal of the reaction, leading ultimately to the formation of acetyl-thiamin pyrophosphate, which undergoes hydrolysis to acetate.
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D007700:Kinetics; D009243:NAD / Q000494:pharmacology*; D010084:Oxidation-Reduction; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013835:Thiamine Pyrophosphate / Q000494:pharmacology*
D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D000105:Acetyl Coenzyme A; D004228:Dithionitrobenzoic Acid; D005033:Ethylmaleimide; D013835:Thiamine Pyrophosphate