Acetyl Coenzyme A Carboxylase System of Escherichia coli (1974)

Abstract Stoichiometric, isotopic exchange and model reaction studies carried out with the resolved, homogeneous protein components (biotin carboxylase, carboxyltransferase, and biotin-containing carboxyl carrier protein) of the acetyl-CoA carboxylase system from Escherichia coli B show that the half-reactions of acetyl-CoA carboxylation occur on different subunits according to the following sequence: CCP (carboxyl carrier protein)-Biotin + HO-CO2- + ATP (Me2+)/⇌/(BC) CCP-biotin-CO2- + ADP + Pi (1) CCP-biotin-CO2- + acetyl-CoA ⇌/(CT) CCP-biotin + malonyl-CoA (2) Net: acetyl-CoA + HO-CO2- + ADP (Me2+)/⇌ malonyl-CoA + ADP + Pi (3) Biotin carboxylase catalyzes the stoichiometric carboxylation of carboxyl carrier protein in the presence of ATP, Mg2+, and HCO3-; ATP-[14C]ADP exchange; and ATP-32Pi exchange in accordance with Reaction 1; divalent cation, bicarbonate, and carboxyl carrier protein are required for both exchanges. Furthermore, ATP-[14C]ADP exchange requires inorganic phosphate, whereas ATP-32Pi exchange requires ADP. Carboxyltransferase carries out net transcarboxylation from the carboxylated carboxyl carrier protein to acetyl-CoA to form malonyl-CoA (Reaction 2) and in addition, catalyzes carboxyl carrier protein-dependent malonyl CoA-[14C]acetyl-CoA exchange. All of the exchange reactions are inhibited by avidin. The two catalytic components of the E. coli carboxylase system (biotin carboxylase and carboxyltransferase), although devoid of covalently bound biotin, catalyze model reactions with free d-biotin derivatives which account for their respective roles in Reactions 1 and 2 and show that each component possesses a specific binding site for the bicyclic ring of the prosthetic group. This is consistent with the view that the biotinyl prosthetic group acts as a mobile carboxyl carrier which oscillates between the carboxylation and transcarboxylation sites on biotin carboxylase and carboxyltransferase, respectively. The fact that biotin carboxylase also catalyzes rapid biotin-dependent phosphoryl transfer from carbamyl phosphate (an analogue of carbonic-phosphoric anhydride) to ADP to form ATP suggests that the carboxylation of free d-biotin or carboxyl carrier protein may proceed via a carbonic-phosphoric anhydride intermediate. In the phosphoryl transfer reaction, biotin appears to fulfill a conformational requirement of the enzyme rather than to participate in the reaction per se. Derivatives of biotin in which the site of carboxylation, i.e. the 1'-ureido-N (1'-N) is substituted and, therefore, inactive in the carboxylation reaction, support the phosphoryl transfer reaction. Moreover, the Km of biotin carboxylase for free d-biotin is more than two orders of magnitude lower for phosphoryl transfer (1 mm) than for carboxylation (170 mm). Carboxyltransferase, which catalyzes transcarboxylation from malonyl-CoA to free d-biotin or its derivatives, also catalyzes a slow decarboxylation of malonyl-CoA to form acetyl-CoA in the absence of a model carboxyl acceptor. 2-Imidazolidone, an analogue of the ureido ring of biotin, does not serve as an acceptor in the carboxyltransfer reaction, but accelerates malonyl-CoA decarboxylation. Thus, although the transferase per se activates C—C bond cleavage in malonyl-CoA, this process is enhanced by binding the 2-imidazolidone ring at the transcarboxylation site. All of the model reactions exhibit similar requirements for maximal activity with respect to the structural features of the biotin molecule, with one notable exception. In the case of biotin carboxylase, a free unprotonated side chain carboxyl group is required for maximal activity while the opposite is true for the carboxyltransferase-catalyzed reaction.