Acetyl coenzyme A carboxylase. IV. Biotinyl prosthetic group-independent malonyl coenzyme A decarboxylation and carbosyl transfer: generalization to other biotin enzymes.

Liver acetyl-CoA carboxylase, a biotin-enzyme which catalyzes the ATP-dependent carboxylation of acetyl-CoA (acceptor) to form malonyl-CoA (carboxylated acceptor), decarboxylates malonyl-CoA by a biotin-dependent, as well as a biotin-independent mechanism. Neither ADP, Pi, nor divalent metal ion are required for either of these abortive decarboxylations. The biotin-dependent reaction is blocked by avidin and is activated synergistically by acetyl-CoA and citrate, an allosteric effector of the carboxylase. However, the biotin-independent process is unaffected by avidin and citrate, and is inhibited slightly by acetyl-CoA. The occurrence of this abortive reaction indicates that the enzyme promotes the labilization of the free carboxyl group of malonyl-CoA and that insertion of a proton in its place is possible without the participation of the biotinyl prosthetic group. This phenomenon appears to be a property of biotin-dependent carboxylases since pig heart propionyl-CoA carboxylase, the biotin-free carboxyltransferase component of Escherichia coli acetyl-CoA carboxylase, and avian liver pyruvate carboxylase catalyze similar biotin-independent decarboxylations of their respective carboxylated acceptor substrates. Liver acetyl-CoA carboxylase also catalyzes carboxyl transfer from [3-14C]malonyl-CoA to free d-biotin, a model for the second half-reaction in the over-all carboxylation process. The acid-labile radioactive carboxylation product was stabilized by methylation with diazomethane, and the ester formed identified as l’-N-carboxy-d-biotin dimethyl ester by carrier recrystallization with the authentic compound. The model reaction does not involve the covalently bound biotiny1 prosthetic group, since the essentially irreversible complex formation of this group with avidin does not block carboxyl transfer. While the reaction is specific in that I-biotin and imidazolidone-2 are inactive, certain closely related biotin derivatives including d-homobiotin and biocytin (e-N-d-biotinyl-L-lysine) are somewhat better acceptor substrates than d-biotin. The fact that citrate blocks carboxy1 transfer to free d-biotin and biocytin with both avidincomplexed and native carboxylase without affecting the rate of malonyl-CoA decarboxylation is compatible with earlier evidence implicating the biotinyl prosthetic group as the focal point of citrate-induced conformational changes.