The elementary reactions of the pig heart pyruvate dehydrogenase complex. A study of the inhibition by phosphorylation.
1. A method was devised for preparing pig heart pyruvate dehydrogenase free of thiamin pyrophosphate (TPP), permitting studies of the binding of [35S]TPP to pyruvate dehydrogenase and pyruvate dehydrogenase phosphate. The Kd of TPP for pyruvate dehydrogenase was in the range 6.2-8.2 muM, whereas that for pyruvate dehydrogenase phosphate was approximately 15 muM; both forms of the complex contained about the same total number of binding sites (500 pmol/unit of enzyme). EDTA completely inhibited binding of TPP; sodium pyrophosphate, adenylyl imidodiphosphate and GTP, which are inhibitors (competitive with TPP) of the overall pyruvate dehydrogenase reaction, did not appreciably affect TPP binding. 2. Initial-velocity patterns of the overall pyruvate dehydrogenase reaction obtained with varying TPP, CoA and NAD+ concentrations at a fixed pyruvate concentration were consistent with a sequential three-site Ping Pong mechanism; in the presence of oxaloacetate and citrate synthase to remove acetyl-CoA (an inhibitor of the overall reaction) the values of Km for NAD+ and CoA were 53+/- 5 muM and 1.9+/-0.2 muM respectively. Initial-velocity patterns observed with varying TPP concentrations at various fixed concentrations of pyruvate were indicative of either a compulsory order of addition of substrates to form a ternary complex (pyruvate-Enz-TPP) or a random-sequence mechanism in which interconversion of ternary intermediates is rate-limiting; values of Km for pyruvate and TPP were 25+/-4 muM and 50+/-10 nM respectively. The Kia-TPP (the dissociation constant for Enz-TPP complex calculated from kinetic plots) was close to the value of Kd-TPP (determined by direct binding studies). 3. Inhibition of the overall pyruvate dehydrogenase reaction by pyrophosphate was mixed non-competitive versus pyruvate and competitive versus TPP; however, pyrophosphate did not alter the calculated value for Kia-TPP, consistent with the lack of effect of pyrophosphate on the Kd for TPP. 4. Pyruvate dehydrogenase catalysed a TPP-dependent production of 14CO2 from [1-14C]pyruvate in the absence of NAD+ and CoA at approximately 0.35% of the overall reaction rate; this was substantially inhibited by phosphorylation of the enzyme both in the presence and absence of acetaldehyde (which stimulates the rate of 14CO2 production two- or three-fold). 5. Pyruvate dehydrogenase catalysed a partial back-reaction in the presence of TPP, acetyl-CoA and NADH. The Km for TPP was 4.1+/-0.5 muM. The partial back-reaction was stimulated by acetaldehyde, inhibited by pyrophosphate and abolished by phosphorylation. 6. Formation of enzyme-bound [14C]acetylhydrolipoate from [3-14C]pyruvate but not from [1-14C]acetyl-CoA was inhibited by phosphorylation. Phosphorylation also substantially inhibited the transfer of [14C]acetyl groups from enzyme-bound [14C]acetylhydrolipoate to TPP in the presence of NADH. 7...
D000079:Acetaldehyde / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001151:Arsenic / Q000494:pharmacology; D003065:Coenzyme A / Q000378:metabolism; D011756:Diphosphates / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D011756:Diphosphates; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D000266:Adenylyl Imidodiphosphate; D008063:Thioctic Acid; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D000079:Acetaldehyde; D001151:Arsenic; D013835:Thiamine Pyrophosphate; D003065:Coenzyme A