Acetyl Coenzyme A Carboxylase System of Escherichia coli

Abstract

The three protein components (biotin carboxylase, carboxyltransferase, and the biotin-containing carboxyl carrier protein of the acetyl coenzyme A carboxylase system have been resolved and purified extensively or to homogeneity from cell-free extracts of Escherichia coli B. Carboxylation of acetyl-CoA to form malonyl-CoA requires the presence of all three components. Biotin carboxylase, which catalyzes the first half-reaction, CCP (carboxyl carrier protein)-biotin + HCO3- + (Me2+)/⇌ CCP-biotin-CO2- + ADP + Pi, (I) has been purified to a homogeneous state and has been crystallized; earlier work in this laboratory showed the enzyme to be composed of two 50,000-dalton subunits. The carboxylation of free d-biotin which can substitute for carboxyl carrier protein-biotin as model substrate is markedly activated by certain organic solvents; an optimal rate enhancement of 10-fold is obtained with 15 % (v/v) ethanol. Activation by ethanol affects Vmax and is not accompanied by changes in Km values or the state of aggregation of the enzyme. Moreover, none of the carboxyl carrier protein-dependent reactions catalyzed by biotin carboxylase, e.g. acetyl-CoA carboxylation, ATP-[14C]ADP exchange, and ATP-32Pi exchange, are activated by organic solvents. Carboxyltransferase, the catalyst for the second half-reaction, CCP-biotin-CO2- + acetyl-CoA ⇌ CCP-biotin + malonyl-CoA, (II) was purified to apparent homogeneity. Biotin carboxylase, although not required for the second half-reaction as measured by carboxyltransferase- and carboxyl carrier protein-dependent malonyl-CoA-[14C]acetyl-CoA exchange, activates this process indicating the existence of a ternary complex between the the three protein components. In addition to the above reaction (Reaction II), carboxyltransferase catalyzes: (a) net transcarboxylation from malonyl-CoA to free d-biotin derivatives in the absence of biotin carboxylase and carboxyl carrier protein, and (b) a slow biotin-independent decarboxylation of malonyl-CoA. The carboxyltransferase component has a molecular weight of 130,000 and is composed of nonidentical polypeptide chains of 30,000 and 35,000 daltons. Carboxyl carrier protein has been purified extensively by a combination of conventional methods and affinity chromatography with Sepharose-avidin (monomer). Polyacrylamide gel electrophoresis of purified carboxyl carrier protein revealed two major proteins both of which contain biotin and exhibit carboxyl carrier protein activity, i.e. carboxyl carrier protein- and carboxyltransferase-dependent malonyl-CoA-[14C]acetyl-CoA exchange. The two catalytic components of the E. coli acetyl-CoA carboxylase system, biotin carboxylase and carboxyltransferase, are devoid of free or covalently bound biotin yet have the ability to carry out their respective model half-reactions utilizing free d-biotin derivatives in place of carboxyl carrier protein. Thus, in addition to possessing binding sites for their respective substrates, each catalytic component must contain a specific binding site for the biotinyl moiety of carboxyl carrier protein. It is evident that during the over-all sequence (Reactions I + II) the caboxylated biotinyl prosthetic group must undergo translocation from the carboxylation site on biotin carboxylase to the transfer site on carboxyl transferase while remaining attached to carboxyl carrier protein through its 14 A side chain.