Locus of action of acetyl CoA in the biotin-carboxylation reaction of pyruvate carboxylase.
The [14C]carboxyphospho-enzyme complex formed by incubation of the enzyme with H14CO3-, MgATP, and Mg2+ was prepared and isolated by gel filtration as described by Phillips et al. [(1992) Biochemistry 31, 9445-9450]. When time courses of transfer of the [14C]carboxyl group from the complex to pyruvate were studied, it was found that at the first time point (15 s) the formation of [14C]oxalacetate was the same in the presence or absence of acetyl CoA. However, in the absence of acetyl CoA, the radioactivity fixed in [14C]oxalacetate declined rapidly over the subsequent 15 min, whereas in the presence of acetyl CoA the formation of [14C]oxalacetate continued up to about 10 min. The decline in [14C]oxalacetate in the absence of acetyl CoA was found to be due to enzyme-dependent decarboxylation of the oxalacetate by the enzyme. Incubation of the isolated [14C]carboxyphospho-enzyme complex with MgADP and Mg2+ resulted in no significant reduction in the formation of [14C]oxalacetate on addition of acetyl CoA and pyruvate. Incubation of the isolated [32P]carboxyphospho-enzyme complex with pyruvate resulted in no significant reduction in the formation of [gamma-32P]ATP on the addition of MgADP and Mg2+. This new evidence casts doubt on the suggested locus of activation of the enzyme by acetyl CoA being the facilitation of the transfer of the carboxyl group from carboxyphosphate to biotin and indeed on the identity of the isolated enzyme intermediate [Phillips et al. (1992) Biochemistry 31, 9445-9450].
Attwood
P V
PV
Department of Biochemistry, University of Western Australia, Nedlands.
D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D001360:Avidin / Q000494:pharmacology; D001710:Biotin / Q000378:metabolism*; D002264:Carboxylic Acids / Q000378:metabolism; D002645:Chickens; D003653:Decarboxylation; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D008099:Liver / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D008293:Malates / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism; D017875:Phosphotransferases (Phosphomutases) / Q000302:isolation & purification / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000378:metabolism*; D011773:Pyruvates / Q000378:metabolism
D002264:Carboxylic Acids; D008293:Malates; D010071:Oxaloacetates; D011773:Pyruvates; D001360:Avidin; D000244:Adenosine Diphosphate; D001710:Biotin; D000105:Acetyl Coenzyme A; D004492:Edetic Acid; D017875:Phosphotransferases (Phosphomutases); C064363:carboxyphosphonoenolpyruvate phosphonomutase; D011766:Pyruvate Carboxylase; D008274:Magnesium