On the mode of action of lipid-lowering agents. 3. Kinetics of activation and inhibition of acetyl coenzyme A carboxylase.

Abstract Rat liver acetyl coenzyme A carboxylase was purified about 200-fold and the inhibition of this enzyme by certain hypolipidemic drugs was studied. The inhibition was more pronounced if the drugs were added before rather than after the citrate activation of the enzyme. Kinetic analysis revealed noncompetitive inhibition of the drugs with respect to the substrates acetyl-CoA, ATP, and HCO3-, and competitive inhibition with respect to the activator, citrate. Sucrose density gradient centrifugations showed that the drugs reverse the aggregating effect of citrate to form the active polymeric forms of the enzyme from the inactive monomers. Arrhenius plots and heat-inactivation studies suggest gross conformational changes of the enzyme protein in the presence of the drugs. Relative affinities of acetyl coenzyme A carboxylase for citrate and the drugs are expressed by the calculated dissociation constant for citrate and the inhibition constants of the drugs. Derangement of fatty acid synthesis in vivo is conceivable by competition of the drugs with the activation of acetyl-CoA carboxylase at low and physiologically possible concentrations of the drugs and citrate.

Kinetic analyses were carried out for the isocitrate activation, and the inhibition by hypolipidemic drugs of avian liver acetyl-CoA carboxylase. Inhibition was found to be competitive for both the substrate acetyl-CoA, and the activator isocitrate and noncompetitive for ATP and HCO,. The isocitrate activation of the enzyme was shown to be due to an elevation of V, values for the substrates, acetyl-CoA and ATP, rather than their apparent Michaelis constants (K,). Molecular orders of participation for acetyl-CoA in the carboxylation reaction, and for isocitrate in the activation process of the enzyme seem to be approximately 1 as shown by Hill-type analysis. Relative affinities of acetyl-CoA carboxylase for acetyl-CoA, isocitrate, and the drugs are expressed by the dissociation constants calculated for acetyl-CoA and isocitrate and the inhibition constants of the drugs. These constants indicate that the drugs may interfere with acetyl-CoA carboxylase activity in vivo either by competing with the activator isocitrate or the substrate acetyl-CoA.
It has been suggested that interaction with thip enzyme, catalyzing the rate-limiting step in lipid biosynthesis (12, 13), could alter the rate and extent of lipogenesis by making malonyl-CoA unavailable for the biosynthetic process.
Experiments with TPIA'-treated animals have shown that both acetyl-CoA carboxylase levels and lipogenesis from [1-14C]acetate in viva are significantly depressed in the drug-treated animals as compared with controls.
sponding to the reduction in total carcass lipids (8). The present paper deals with the Dixon plots of the inhibition of avian liver acetyl-CoA carboxylase by CPIB and TPIA and the kinetics of the activation of this enzyme by isocitrate.
Kinetic constants and relative affinities of acetyl-CoA, isocitrate, and drugs with the enzyme are discussed in relation to the mechanism of the inhibition.

MATERIALS AND METHODS
ATP, acetyl-CoA, and glutathione were obtained from Sigma Chemical Company. dl-Isocitric acid, trisodium salt, was obtained from J. T. Baker Chemical Company, Philipsburg, New Jersey, and [14C]-HC0s-was purchased from New England Nuclear Company, Boston, Massachusetts.
CPIB is a product of Imperial Chemical Industries, England. TPIA is an experimental hypolipidemic compound synthesized in our laboratories by Dr. W. Bencze.
The reaction mixture, usually 0.5 ml contained in an ampoule, was placed into a liquid scintillation vial, was briefly exposed to gaseous HCl, and dried under a stream of warm air at 40-50".
The HCl treatment ensures complete release of all unreacted [14C]-C02. Alcohol and scintillat,ion mixture were added and the Y-activity was determined by the use of a liquid scintillation spectrometer (TracerLab, Waltham, Massachusetts) under flat spectrum counting conditions (15). The reaction follows zero order kinetics for at least 11 min under the assay conditions. Issue of August 25, 1970 M, E. ibfaragoudakis 4137 of these compounds.
Therefore, a detailed kinetic study of the inhibition was made to probe its mechanism. We have shown by Lineweaver-Burk plots (16) that the inhibition is competitive for isocitrate and acetyl-CoA and noncompetitive for ATP and HC03. It is difficult to visualize how these drugs can compete with both acetyl-CoA and isocitrate for their respective binding sites on the enzyme protein.
Palmitoyl-CoA, another known inhibitor of acetyl-CoA carboxylase (17), is competitive for citrate but noncompetitive for acetyl-CoA.
The binding sites, therefore, for isocitrate and acetyl-CoA must be independent so that palmitoyl-CoA can bind to one without an apparent effect on the other.  Table I. petition of CPIB and TPIA with both acetyl-CoA and isocitrate suggests that the drugs bind either independently to the sites for acetyl-CoA and isocitrate or to a third site located in such a position on the enzyme protein that inhibitors bound there would affect the binding affinities of both acetyl-CoA and isocitrate.
We felt that kinetic analysis by an independent method should be done in order to confirm the competition of CPIB and TPIA with the unrelated molecular structures of acetyl-CoA and isocitrate for their enzyme-binding sites. Figs. 1 through 4 show the Dixon plots for acetyl-CoA, ATP, isocitrate, and bicarbonate when TPIA is used as inhibitor (18)   is noncompetitive for ATP and bicarbonate and competitive for isocitrate.
The same results were obtained with CPIB. The kinetic constants calculated from these data are compiled in Table I. A close agreement of the inhibition constants obtained by Lineweaver-Burk and Dixon plots is evident. Mechanism of Isocitrate Activation-The kinetics of the citrate activation of rat liver acetyl-CoA carboxylase have been studied by Numa, Ringelmann, and Lynen (17). Gregolin, Ryder, and Lane (14) have studied the activation of avian liver acetyl-CoA carboxylase by isocitrate, but they were able to obtain linear Lineweaver-Burk plots only when acetyl-CoA was the varying substrate.
In view of the interference of CPIB and TPIA with the activation process of acetyl-CoA carboxylase, it seemed desirable to study in some detail the kinetics of the isocitrate-activation. The steady state kinetic parameters were obtained from the rate data by means of a digital computer program written for the Lineweaver-Burk form of the Michaelis-Menten rate equation. Data weighting and calculation of standard errors of the kinetic constants were performed by the method of Wilkinson (19). Table II shows the kinetic constants obtained from double reciprocal plots for variable concentration of acetyl-CoA at three levels of isocitrate concentration.
From these data it is evident that the isocitrate-activation of avian liver acetyl-CoA carboxylase is caused by an increase of the V,,, rather than the K, value for acetyl-CoA.
The same results were obtained at levels of isocitrate 1.25, 2.5, and 5 mM (data not shown).
Allosteric effecters which elicit V, changes (20) can be described kinetically (21) by the expression (1) where V = reaction rate, K' = rate constant, e = total enzyme, X = substrate concentration, a = activator concentration, and K, and K, are the dissociation constants for substrate and activator, respectively.
This equation describes the independent combination of activator and substrate with enzyme. Variation of either a or S with fixed level of the other will give a common K, or K,, but a different V,,, for each level of the fixed one of the two changing variables. Independent combination of acetyl-CoA carboxylase with acetyl-CoA and isocitrate was also suggested from data obtained by varying isocitrate concentration at three levels of acetyl-Co-4. K, for isocitrate remained constant, but V,, was changed as predicted from Equation 1. (Data not shown.) Table II summarizes the kinetic constants of the Lineweaver-Burk plots for varying ATP concentration at levels of isocitrate 0.625, 1.25, and 2 mm. At 0.625 and 1.25 mM of isocitrate the effect seems to be on V,,, only. At higher isocitrate levels (2 rnM) both K,and V,,,are affected. Numa et al. (17) have obtained the same results at the higher citrate levels with the rat enzyme.
Table II also suggests that activation of acetyl-CoA carboxylase is associated primarily with a V,,, effect for varying isocitrate concentrations at four concentration levels of ATP. Kinetic data for HC03 are summarized in Table II. Molecular Or& of Pa&tip&ion-In order to obtain information as to whether one or more isocitrate molecules per enzyme molecule is involved in the activation of acetyl-CoA carboxylase, the data of the activity of the enzyme at varying isocitrate con- From the experimental data, the apparent dissociation constant (K) can be calculated by measuring log (VIVnmx -V) at any value of log S and substituting the value of n = 1.03, or taking the value of the intercept given by the computer.
It was thus found that K = [(enzyme)(isocitrate)]/ (enzyme -isocitrate) = 1.34 X 10e3 M. The activation of acetyl-CoA carboxylase by isocitrate, therefore, is taking place by interaction of 1 molecule of isocitrate with 1 molecule of enzyme to form a complex with an apparent dissociation constant K = 1.34 X lo+ M. Fig. 6 shows a plot of log (V/V,,, -V) against thelog of the acetyl-CoA concentration. The Hill constant (n) obtained for acetyl-CoA is n = 1.07 f 0.02 and the apparent dissociation constant calculated for the enzyme-acetyl-Cob complex is K = 7.8 X 10-5M.
Values of the apparent dissociation constants for acetyl-CoA and isocitrate indicate that acetyl-CoA has greater affinity for the enzyme than does isocitrate.
Inhibition constants of the drugs obtained from the kinetic analysis are true dissociation constants of the drug-enzyme complexes.
The relative affinities therefore of drugs, acetyl-CoA, and isocitrate for the enzyme can be calculated from the ratios of K/Ki.
With respect to isocitrate, this ratio is found to be K/K; = 5.6 for CPIB and K/Ki = 13.4 for TPIA.
With respect to acetyl-CoA K/K; = 6.5 x 10e2 for CPIB and K/Ki = 0.84 for TPIA.  (23), and from these we have calculated the difference in the free energy of binding of acetyl-CoA and CPIB, or TPIA, with the enzyme. This difference in free energy of binding of acetyl-CoA and TPIA with acetyl-CoA carboxylase is AF = 107 cal per mole. The same parameter for CPIB is AF = 4500 cal per mole. Similarly the difference in free energy of binding of isocitrate and TPIA or CPIB with acetyl-CoA carboxylase is AF = -1600 cal per mole for TPIA and AF = -1050 cal per mole for CPIB. DISCUSSION The critical importance of the carboxylation of acetyl-Co-4 to malonyl-CoA in fatty acid biosynthesis has long been recognized. Evidence from many laboratories (12, 13) points to this reaction as the rate-limiting step of the over-all conversion of acetyl-CoA to fatty acids by the liver. In metabolic conditions in which fatty acid synthesis is depressed, the locus of this depression effect has been shown to be the carboxylation reaction (24, 25).
In previous reports (7-111, we have studied the inhibitory effect of certain lipid-lowering agents on acetyl-CoA carboxylase purified from avian or rat liver. This inhibition is viewed as a possible mechanism of action of these drugs. As shown by kinetic analysis, interaction of acetyl-CoA carboxylase with the hypolipidemic compounds studied is taking place by competition of the drugs with acetyl-CoA or isocitrate for the same enzyme site. It is uncertain whether interference with the activation of the enzyme or competition at the substrate level is more important in the over-all inhibitory effect of acetyl-CoA carboxylase by CPIB and TPIA.
From the apparent dissociation constants of the enzyme complexes with TPIA, CPIB, isocitrate, and acetyl-CoA, it appears that each one of these molecules can form a complex with the enzyme protein with relative order of stability, acetyl-CoA > TPIA > CPIB > isocitrate. TPIA and CPIB can displace acetyl-CoA from the enzyme-