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Acetyl coenzyme A carboxylase has been isolated from chicken liver and purified more than 1000-fold in good yield. It is an unusually stable protein exhibiting negligible loss of enzymatic activity during 1 week of storage (1 to 5 mg of protein per ml) at room temperature in potassium phosphate buffer, pH 7. The pure enzyme catalyzes the carboxylation of 8.8 µmoles of acetyl-CoA (or 7.0 µmoles of propionyl-CoA) per min per mg of refractometrically determined protein at its pH optimum of 7.5 at 37°. The carboxylase-catalyzed carboxylation of acetyl-CoA is activated 15- to 16-fold by dl-isocitrate or citrate and 5-fold by malonate; tricarballylate is essentially inactive. Arrhenius plots for acetyl-CoA carboxylation in the presence or absence of isocitrate are biphasic, having a transition point at about 21°. Below this temperature, where there is very little activator effect, the temperature coefficient (Q10) in the presence of isocitrate is 8.0. Above the transition point, where isocitrate activation is evident, the temperature coefficient (Q10) in the presence of activator is 2.0 and in its absence is a negative value. At pH 7.5 and 37°, the carboxylase catalyzes the over-all reverse reaction (decarboxylation), ATP-32Pi exchange, malonyl-CoA-14C-acetyl-CoA exchange, and ADP-, Pi-, and Mg2+-independent malonyl-CoA decarboxylation reactions at 14%, 6.2%, 33%, and 1.2%, respectively, of the rate of the over-all forward (carboxylation) reaction. All of the above mentioned reactions exhibit essentially the same tri- and dicarboxylic acid activation patterns. Evidence is presented which indicates that isocitrate activation of the carboxylase is associated with an increased maximum velocity.

The binding of Mn2+ and substrates to phosphoenolpyruvate carboxykinase has been studied by four methods: equilibrium dialysis, gel filtration, electron paramagnetic resonance, and the proton relaxation rate of water (PRR). A binary enzyme-Mn2+ complex was detected by electron paramagnetic resonance and PRR. Binary enzyme-substrate complexes were detected for guanosine triphosphate and guanosine diphosphate by equilibrium dialysis and for inosine diphosphate by gel filtration. Ternary complexes containing enzyme, Mn2+, and substrate were detected for IDP by gel filtration and PRR, for phosphoenolpyruvate by equilibrium dialysis and PRR, and for inosine triphosphate and oxalacetate by PRR. Higher order complexes of the form eMn-sMn were detected for GDP and GTP by equilibrium dialysis. Phospholactate, an analogue of phosphoenolpyruvate, formed ternary complexes with enzyme and Mn2+ and a stable quaternary complex with enzyme, Mn2+, and IDP, as detected by PRR. Where comparable, the dissociation constants obtained by the various methods were in good agreement. The enzyme enhanced the effect of Mn2+ on the PRR of water (eb = 14.2), and this enhancement was reduced in the ternary complexes with phosphoenolpyruvate (et = 6.7) and with IDP (et = 10.4). Since the relaxation mechanism was the same for each of these complexes (measuring the rate of chemical exchange of water protons into the coordination sphere of Mn2+), the relative values of e are consistent with the formation of enzyme-metal-substrate bridge complexes. The related enzyme, phosphoenolpyruvate carboxylase, gives similar results, forming a binary enzyme-Mn2+ complex (eb = 13.8) and a ternary complex with phosphoenolpyruvate (et ≤ 11.3). The Kd of the binary complex agrees with the activator constant for Mn2+ obtained kinetically. The complexes formed by both carboxylating enzymes are thus similar to those found previously for muscle pyruvate kinase and, therefore, suggest homologous mechanisms for phosphoenolpyruvate carboxykinase, phosphoenolpyruvate carboxylase, and pyruvate kinase.

The factors effecting the reversible interconversion between the protomeric and polymeric forms of liver acetyl coenzyme A carboxylase were investigated by sucrose density gradient centrifugation and electron microscopy. Certain anions (citrate, isocitrate, malonate, tricarballylate, sulfate, and Pi), acetyl-CoA, high protein concentration, and pH 6 to 7 promote aggregation of the protomer. Other factors, such as carboxylation of the enzyme to produce enzyme-CO2-, Cl-, and pH values greater than 7.5, cause dissociation of the polymeric form. Citrate and isocitrate, but not tricarballylate or Pi, are capable of promoting the transition of the carboxylated enzyme (enzyme-CO2-) to the polymeric state. Under the conditions for carboxylase assay or for measuring the rates of other carboxylase-catalyzed reactions, all of which involve enzyme-CO2- as intermediate, isocitrate and citrate promote aggregation of the carboxylase to its polymeric form and a 15- to 16-fold activation of catalysis; neither tricarballylate nor Pi has this effect. Binding experiments reveal that acetyl-CoA carboxylase has one tight binding site per protomer (mol wt 410,000) for citrate (Kd, 2 to 3 x 10-6 m) and another for acetyl-CoA (Kd, 4 x 10-6 m). Acetyl-CoA binding at the latter site is unaffected by the presence of citrate. Dissociation of acetyl-CoA carboxylase with sodium dodecyl sulfate (SDS; 0.1%) gives rise to subunits with a sedimentation coefficient (s20,w) of 4.3 S. By equilibrium dialysis against 0.1% SDS-35S, the carboxylase was found to bind 0.57 g of the detergent per g of carboxylase protein. The subunit molecular weight, determined by sedimentation equilibrium corrected for detergent binding, was 114,000 and by electrophoresis in SDS-polyacrylamide gels (1% SDS), 110,000. The presence of nonidentical subunits is indicated by the fact that there is a single biotinyl prosthetic group and there are single binding sites for both citrate and acetyl-CoA on the 410,000 molecular weight protomer.

Spinach leaf ribulose 1,5-diphosphate carboxylase is reversibly inhibited by cyanide at low concentration; 10-5 m and 10-4 m cyanide inhibit the carboxylation reaction 51% and 91%, respectively. Inhibition by cyanide (Ki = 1.6 x 10-5 m) is uncompetitive (anticompetitive) with respect to ribulose 1,5-diphosphate (ribulose-1,5-di-P) and is of mixed character with respect to Mg2+ and HCO3-. This and other kinetic evidence suggest that cyanide combines readily with enzyme-ribulose-1,5-di-P complex, but not with enzyme. The inference that enzyme-ribulose-1,5-di-P complex reacts with cyanide to form an inactive ternary complex is supported by binding studies with the use of the gel filtration technique. Ribulose-1,5-di-P uniformly labeled with 14C is tightly bound to the carboxylase (1.04 moles of ribulose-1,5-di-P per mole of enzyme) only in the presence of cyanide and 14C-cyanide is bound to the carboxylase (1.06 moles of cyanide per mole of enzyme) only in the presence of ribulose-1,5-di-P. The presence of Mg2+ is without effect on 14C-ribulose-1,5-di-P binding in the presence of cyanide or on 14C-cyanide binding in the presence of ribulose-1,5-di-P under the conditions used. A ternary complex of ribulose-1,5-di-P carboxylase, ribulose-1,5-di-P, and cyanide in a mole ratio of 1:1:1 is indicated. Attempts to demonstrate Schiff base formation between the carboxylase and ribulose-1,5-di-P under a wide variety of conditions were unsuccessful.

The active species of "CO2", i.e. CO2 or HCO3-, utilized in the ribulose diphosphate carboxylase reaction was determined with the use of homogeneous preparations of the spinach enzyme. Employing an assay system predicated upon the observation that the hydration of CO2 requires more than 60 sec to reach equilibrium at temperatures below 15°, data were obtained which are quite compatible with those expected if CO2 is the active species.

Recent investigations in this laboratory have shownl, 2 that the isocitrate(or citrate-) activated form of liver acetyl CoA carboxylase (E.C. 6.4.1.2) is a large protein structure having a molecular weight of about four million. Electron microscopic examination of the carboxylase in the presence of isocitrate reveals' that it has a filamentous structure with dimensions of 80-100 A by up to 5000 A. Reversible dissociation to inactive protomeric subunits (molecular weight 409,000) occurs2 at low enzyme concentration in the carboxylation assay reaction mixture in the absence of isocitrate or at higher enzyme concentration in the presence of 0.5 M NaCl at pH 8.0. Reconstitution to the active filamentous form can be accomplished either by addition of isocitrate (or citrate) or by removal of NaCl and introduction of isocitrate by dialysis. The carboxylation of acetyl CoA catalyzed by liver acetyl CoA carboxylase involves the following two partial reactions (reactions 1 and 2)