Effects of adenosine phosphates and nicotinamide nucleotides on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8.0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD(+), NADP(+) and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD(+), NADH, NADP(+) and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6.5, in the presence of 12mm-Mg(2+), 0.75mm-Mn(2+) and 0.5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.     

Synthesis of pyruvate carboxylase from its apoenzyme and (+)-biotin in Bacillus stearothermophilus. Purification and properties of the apoenzyme and the holoenzyme synthetase

1. Methods are described for the assay and purification of pyruvate apocarboxylase and pyruvate holocarboxylase synthetase from biotin-deficient Bacillus stearothermophilus. 2. Pyruvate apocarboxylase was obtained 200-fold purified and in a nearly homogeneous state; it closely resembled the holoenzyme of the thermophile in fractionation properties, electrophoretic mobility and molecular weight (estimated to be 350000 by gel filtration). 3. Pyruvate holocarboxylase synthetase, purified more than 50-fold, was estimated to have a molecular weight of approx. 40000. 4. The conversion of the purified apoenzyme into the holoenzyme required the presence of the synthetase, ATP (K_m3.3×10⁻⁷m), (+)-biotin (K_m7.5×10⁻⁸m) and Mg²⁺; it differed from the conversions effected by systems forming other carboxylases in mesophilic organisms in also requiring the presence of acetyl-CoA.     

Kinetic characterization of yeast pyruvate carboxylase isozyme Pyc1 and the Pyc1 mutant, C249A

The yeast Pyc1 isoform of pyruvate carboxylase has been further characterized and shown to differ from the Pyc2 isoform in its K(a) for K(+) activation. Pyc1 differs from chicken liver pyruvate carboxylase in the lack of effect of acetyl-CoA on ADP phosphorylation by carbamoyl phosphate, which may be a result of differences in the loci of action of the effector between the two enzymes. Solvent D(2)O isotope effects have been measured with Pyc1 on the full pyruvate carboxylation reaction, the ATPase reaction in the absence of pyruvate, and the carbamoyl phosphate-ADP phosphorylation reaction for the first time for pyruvate carboxylase. Proton inventories indicate that the measured isotope effects are due to a single proton transfer step in the reaction. The inverse isotope effects observed in all reactions suggest that the proton transfer step converts the enzyme from an inactive to an active form. Kinetic measurements on the C249A mutant enzyme suggest that C249 is involved in the binding and action of enzyme activators K(+) and acetyl-CoA. C249 is not involved in ATP binding as was observed for the corresponding residue in the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase, nor is it directly responsible for the measured inverse (D)(k(cat)/K(m)) isotope effects. The size of the inverse isotope effects indicates that they may result from formation of a low-barrier hydrogen bond. Modification of the wild type and C249A mutant with o-phthalaldehyde suggests that C249 is involved in isoindole formation but that the modification of this residue is not directly responsible for the accompanying major loss of enzyme activity.     

GroE dependence of refolding and holoenzyme formation of 6-hydroxy-D-nicotine oxidase.

In Escherichia coli cells expressing 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoprotein with covalently bound FAD, approximately 40% of the polypeptide is in its apoform. We investigated whether in vivo holoenzyme formation was influenced by the association of the apoenzyme with cellular chaperones. Immunoprecipitation of apoenzyme-containing cell extract with protein-A-Sepharose-bound 6-HDNO- or GroEL-specific antibodies failed to reveal the formation of complexes between these proteins. The limiting factor in holoenzyme formation in vivo appeared to be the intracellular supply of phosphorylated tricarbon compounds (e.g. glycerol-3-P) acting as allosteric effectors in the flavinylation reaction. When holoenzyme formation from purified apo6-HDNO was investigated in vitro, addition of GroEL and GroES to the reaction assays increased the yield of holoenzyme formation. The observed increase in apoenzyme to holoenzyme transition was ATP independent, and the effect of GroE could be simulated by high concentrations of glycerol (40%). Apparently, a nonspecific protein-protein interaction between the GroE proteins and the apo6-HDNO favored holoenzyme formation. The refolding of guanidinium hydrochloride-unfolded holoenzyme, however, was catalyzed by GroEL and GroES in an ATP-dependent reaction. Recovery of the native, enzymatically active, conformation ranged from 30 to 40%. When apo6-HDNO was denatured and refolded, the same dependence on GroE and ATP was observed in the recovery of a conformation able to incorporate FAD and to holoenzyme. [14C] FAD in the refolding assay yielded radioactively labeled 6-HDNO demonstrating the autocatalytical covalent incorporation of FAD into the polypeptide during the folding process.     

Pig liver pyruvate carboxylase. The reaction pathway for the carboxylation of pyruvate

1. The reaction pathway for the carboxylation of pyruvate, catalysed by pig liver pyruvate carboxylase, was studied in the presence of saturating concentrations of K(+) and acetyl-CoA. 2. Free Mg(2+) binds to the enzyme in an equilibrium fashion and remains bound during all further catalytic cycles. MgATP(2-) binds next, followed by HCO(3) (-) and then pyruvate. Oxaloacetate is released before the random release, at equilibrium, of P(i) and MgADP(-). 3. This reaction pathway is compared with the double displacement (Ping Pong) mechanisms that have previously been described for pyruvate carboxylases from other sources. The reaction pathway proposed for the pig liver enzyme is superior in that it shows no kinetic inconsistencies and satisfactorily explains the low rate of the ATP[unk][(32)P]P(i) equilibrium exchange reaction. 4. Values are presented for the stability constants of the magnesium complexes of ATP, ADP, acetyl-CoA, P(i), pyruvate and oxaloacetate.     

The atypical velocity response by pyruvate carboxylase to increasing concentrations of acetyl-coenzyme A

An investigation was made of the interaction of pyruvate carboxylase with its allosteric effector, acetyl-CoA, and the velocity profile of the deacylation of acetyl-CoA as a function of acetyl-CoA concentration indicated that this ligand does not bind to this enzyme in a positive homotropic co-operative manner. An examination was therefore made of the factors that contribute to the sigmoidicity of the rate curves obtained for pyruvate carboxylation with various concentrations of acetyl-CoA. Hill coefficients for acetyl-CoA obtained with both sheep and chicken liver pyruvate carboxylases were found to be dependent on the fixed pyruvate concentration used in the assay solution. Thus, by varying the acetyl-CoA concentration, the degree of saturation of the enzyme by pyruvate was also changed. A further consequence of non-saturating concentrations of pyruvate was that the non-productive hydrolysis of the enzyme- carboxybiotin complex increased, resulting in an under-estimate of the reaction velocity measured by oxaloacetate formation. Another factor contributing to the sigmoidicity is that, at non-saturating concentrations of acetyl-CoA, the enzyme undergoes inactivation upon dilution to low protein concentrations, again resulting in an under-estimate of the reaction velocity. Under conditions where none of the above factors was operating and the only effect of varying acetyl-CoA concentrations was to alter the proportion of the enzyme catalysing the carboxylation reaction at acetyl-CoA-dependent and -independent rates, the sigmoidicity of the acetyl-CoA velocity profile was completely eliminated.     

Regulation of fatty acid synthesis.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.     

Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes

The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.     

Acetyl-Coenzyme A carboxylase. Role of the prosthetic group in enzyme polymerization.

Apo-(acetyl-CoA carboxylase) completely free from the holoenzyme was prepared from biotin-deficient rat adipose tissue by using affinity chromatography. The apoenzyme does not aggregate under conditions favouring the transition of the holoenzyme to the polymeric form. Such transition is possible after the conversion of the apoenzyme into the holoenzyme in vitro, thus demonstrating the requirement of the prosthetic biotinyl group for enzyme activation.     

Enzyme systems of the substrate and cofactor supply of hyperlipogenesis in non-insulin-dependent diabetes

Enzymatic systems of hepatic hyperlipogenesis supply by substrate (acetyl-CoA) and cofactors (NADPH and ATP) were studied in experiments on diabetic C57Bl/Ks J mice (db/db) that served as a model of non-insulin dependent diabetes. The rise in acetyl-CoA synthetase activity catalyzing the primary step of lipogenesis from acetate has been found, while pyruvate dehydrogenase complex activity did not differ from the control and ATP-citrate lyase activity was lowered. Hyperlipogenesis in non-insulin dependent diabetes was induced by the activation of cellular energy supply revealed in enhanced 2-oxoglutarate dehydrogenase activity and elevated ATP level, as well as changes in the activity ratio of NADPH supply and utilization and the rise in fructose-1,6-diphosphate, allosteric effector of fatty acid synthetase, which resulted in the increase of the enzyme activity and created wider potentials of NADPH utilization as a reducing equivalent in lipogenesis.     

Characterization and Regulation of Pyruvate Carboxylase of Bacillus licheniformis

Cell-free extracts of Bacillus licheniformis were found to contain pyruvate carboxylase which catalyzes the reaction between pyruvate and bicarbonate to yield oxalacetate in the presence of adenosine triphosphate (ATP), acetylcoenzyme A (CoA), and manganese. The plot between the reaction velocity of the carboxylation by the partially purified pyruvate carboxylase (25-fold) and the concentration of pyruvate, bicarbonate, manganese, and ATP did not indicate a pronounced deviation from the Michaelis-Menten hyperbola. The enzyme was inhibited by avidin and aspartate. Biotin partially protected the enzyme from avidin inhibition, whereas the amount of inhibition by aspartate was dependent on the concentration of acetyl-CoA present. The intracellular concentration of acetyl-CoA did not vary significantly enough to allow control of the enzyme by this method. Extracts of 4-hr postexponential-phase cells of B. licheniformis were also found to contain phosphoenolpyruvate carboxykinase, which appears to be under catabolite repression control. It is suggested that the endogenous induction of this enzyme is the determining factor allowing the shift to gluconeogenesis from glycolysis during sporulation of glucose-grown cells.     

Ligand-induced conformational transitions and secondary-structure composition of chicken liver pyruvate carboxylase

Apparent conformational transitions induced in chicken liver pyruvate carboxylase by substrates, KHCO(3) and MgATP, and the allosteric effector, acetyl-CoA, were studied by using the fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid and c.d. Fluorescence measurements were made with both conventional and stopped-flow spectrophotometers. Additions of acetyl-CoA and/or ATP to the enzyme-probe solutions quenched fluorescence of the probe by the following cumulative amounts regardless of the sequence of additions: acetyl-CoA, 10-13%; ATP, 21-24%; acetyl-CoA plus ATP, about 35%. Additions of KHCO(3) had no effect on the fluorescence. The rates of quenching by acetyl-CoA and MgATP (in the presence of acetyl-CoA) were too rapid to measure by stopped-flow kinetic methods, but kinetics of the MgATP effect (in the absence of acetyl-CoA) indicate three unimolecular transitions after the association step. The negligible effect of the probe on enzyme catalytic activity, a preservation of the near-u.v. c.d. effect of MgATP and acetyl-CoA in the presence of the probe and no observable unimolecular transitions after binding of the probe to the enzyme indicate that the probe had no deleterious effect on the enzyme. In contrast with results with 8-anilinonaphthalene-1-sulphonic acid, fluorescence of the epsilon-derivative of acetyl-CoA or ATP [fluorescent analogues; Secrist, Barrio, Leonard & Weber (1972) Biochemistry11, 3499-3506] was not changed when either one was added to the enzyme. Secondary-structure composition of chicken liver pyruvate carboxylase estimated from the far-u.v. c.d. spectrum of the enzyme is 27% helix, 7% beta-pleated sheet and 66% other structural types.     

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.     

Initiation of lipogenic enzyme activities in rat mammary glands.

The activities of acetyl-CoA carboxylase, ATP citrate-lyase and fatty acid synthetase remained low until parturition at 22 days of gestation and increased significantly within 1 day post partum. Administration of progesterone on days 20 and 21 and at parturition abolished the increases for at least 48 h after parturition. Removal of the pups of normal rats prevented the increases in activities of acetyl-CoA carboxylase and ATP citrate-lyase, but not of fatty acid synthetase, and administration of prolactin corticosterone or insulin did not stimulate activity. Tissue from suckled glands in which the ducts had been ligated at parturition showed no increase in the activities of acetyl-CoA carboxylase and ATP citrate-lyase within 24 h, whereas fatty acid synthetase activity was similar to that in the sham-operated contralateral glands. Foetoplacentectomy on day 18 increased the activity of fatty acid synthetase but not of acetyl-CoA carboxylase and ATP citrate-lyase; suckling of these dams by foster pups increased both acetyl-CoA carboxylase and ATP citrate-lyase.     

Pyruvate carboxylase from a thermophilic Bacillus. Studies on the specificity of activation by acyl derivatives of coenzyme A and on the properties of catalysis in the absence of activator

1. Oxaloacetate synthesis catalysed by pyruvate carboxylase from a thermophilic Bacillus in the absence of acetyl-CoA required addition of high concentrations of pyruvate, MgATP(2-) and HCO(3) (-), and at 45 degrees C occurred at a maximum rate approx. 20% of that in the presence of a saturating concentration of acetyl-CoA. The apparent K(m) for HCO(3) (-) at pH7.8 was 400mm without acetyl-CoA, and 16mm with a saturating activator concentration. The relationship between reciprocal initial rate and reciprocal MgATP(2-) concentration was non-linear (convex-down) in the absence of acetyl-CoA, but the extent of deviation decreased as the activator concentration was increased. The relationship between reciprocal initial rate and reciprocal pyruvate concentration was non-linear (convex-down) in the presence or absence of acetyl-CoA. 2. The optimum pH for catalysis of oxaloacetate synthesis was similar in the presence or absence of acetyl-CoA. The variation with pH of apparent K(m) for HCO(3) (-) implicated residue(s) with pK(a) 8.6 in catalysis of the activator-independent oxaloacetate synthesis. 3. Linear Arrhenius and van't Hoff plots were observed for the temperature-dependence of oxaloacetate synthesis in the absence of acetyl-CoA over the range 25-55 degrees C. E(a) (activation energy) was 56.3kJ/mol and DeltaH(double dagger) (HCO(3) (-)) (enthalpy of activation) was -38.6kJ/mol. In the presence of acetyl-CoA, biphasic Arrhenius and van't Hoff plots are observed with a change of slope at 30 degrees C in each case. E(a) was 43.7 and 106.3kJ/mol above and below 30 degrees C respectively. 4. Incubation of Bacillus pyruvate carboxylase with trinitrobenzenesulphonate caused specific inactivation of acetyl-CoA-dependent catalytic activity associated with the incorporation of 1.3+/-0.2 trinitrophenyl residues per subunit. Activator-independent catalysis and regulatory inhibition by l-aspartate were unaffected. The rate of inactivation of acetyl-CoA-dependent catalysis by trinitrobenzenesulphonate was specifically decreased by addition of acetyl-CoA and other acetyl-CoA and other acyl-CoA species, but complete protection was not obtained. 5. All alkylacyl derivatives of CoA tested activated Bacillus pyruvate carboxylase; acetyl-CoA was the most effective. The apparent K(a) exhibited a biphasic relationship with acyl-chain length for the straight-chain homologues. Certain long-chain acyl-CoA species showed additional activation at a high concentration. Weak activation occurred on addition of CoA or adenosine 3',5'-bisphosphate, but carboxyacyl-CoA species and derivatives containing a modified phosphoadenosyl group were inhibitory. Thioesters of CoA with non-carboxylic acids, e.g. methanesulphonyl-CoA, serve as activators of the thermophilic Bacillus and Saccharomyces cerevisiae pyruvate carboxylases, but as inhibitors of pyruvate carboxylases obtained from chicken and rat liver. 6. alpha-Oxoglutarate mimics the effect of l-aspartate as a regulatory inhibitor of the pyruvate carboxylases from both the thermophilic Bacillus and Saccharomyces cerevisiae. l-Glutamate was ineffective in both cases.     

Densitization of pyruvate carboxylase against acetyl-CoA stimulation by chemical modification

Sheep kidney pyruvate carboxylase has been desensitized against its allosteric effector, acetyl CoA, by limited covalent modification with trinitrobenzene sulphonic acid.Trinitrophenylation of the enzyme resulted in a strong inhibition of the rate of the acetyl CoA-stimulated pyruvate carboxylation and enhancement of the rate of the acetyl CoA-independent reaction. A good correlation was found between the requirement for acetyl CoA of the exchange reactions catalysed by the enzyme and the extent of their inhibition by trinitrobenzene sulphonic acid modification.Spectrophotometric data indicated that one to two lysyl residues per monomer were trinitrophenylated. Modification had only a slight effect on the sedimentation properties of the enzyme.     

The role of pyruvate dehydrogenase and acetyl-coenzyme A synthetase in fatty acid synthesis in developing Arabidopsis seeds

Acetyl-coenzyme A (acetyl-CoA) formed within the plastid is the precursor for the biosynthesis of fatty acids and, through them, a range of important biomolecules. The source of acetyl-CoA in the plastid is not known, but two enzymes are thought to be involved: acetyl-CoA synthetase and plastidic pyruvate dehydrogenase. To determine the importance of these two enzymes in synthesizing acetyl-CoA during lipid accumulation in developing Arabidopsis seeds, we isolated cDNA clones for acetyl-CoA synthetase and for the ptE1alpha- and ptE1beta-subunits of plastidic pyruvate dehydrogenase. To our knowledge, this is the first reported acetyl-CoA synthetase sequence from a plant source. The Arabidopsis acetyl-CoA synthetase preprotein has a calculated mass of 76,678 D, an apparent plastid targeting sequence, and the mature protein is a monomer of 70 to 72 kD. During silique development, the spatial and temporal patterns of the ptE1beta mRNA level are very similar to those of the mRNAs for the plastidic heteromeric acetyl-CoA carboxylase subunits. The pattern of ptE1beta mRNA accumulation strongly correlates with the formation of lipid within the developing embryo. In contrast, the level of mRNA for acetyl-CoA synthetase does not correlate in time and space with lipid accumulation. The highest level of accumulation of the mRNA for acetyl-CoA synthetase during silique development is within the funiculus. These mRNA data suggest a predominant role for plastidic pyruvate dehydrogenase in acetyl-CoA formation during lipid synthesis in seeds.     

Binding of allosteric effectors to carbamyl-phosphate synthetase from Escherichia coli.

The binding of ornithine and inosine 5'-monophosphate (IMP), positive allosteric effectors, and of uridine 5'-monophosphate (UMP), a negative allosteric effector, to carbamyl-phosphate synthetase from Escherichia coli was studied by the technique of equilibrium dialysis. The monomeric form of the enzyme has one binding site for each of the three allosteric ligands. The binding of UMP is inhibited by ornithine, IMP, MgATP, and ammonia (also a positive allosteric effector). Bicarbonate, L-glutamine, and adenosine 5'-triphosphate (ATP) (Mg2+ absent) had no effect on the binding of UMP. The affinity of the enzyme for UMP was increased if phosphate buffer was replaced by 2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffer. The binding of ornithine was inhibited by UMP and ammonia, enhanced by MgATP, MgADP, and IMP, and not affected by bicarbonate, L-glutamine, or ATP (Mg2+ absent). Ornithine and ammonia probably bind to the same site on the enzyme. The binding of IMP is facilitated by ornithine and ammonia, but is inhibited by MgATP or ATP, indicating that adenine nucleotides can also bind to the IMP binding site. The results of these binding studies are consistent with a scheme previously proposed in which the allosteric effectors function by stabilizing one or the other of two different conformational states of the enzyme which are in equilibrium with each other (Anderson, P.M., and Marvin, S.V. (1970), Biochemistry 9, 171). According to this scheme, binding of the substrate MgATP is greatly facilitated when the enzyme exists in the conformational state stabilized by the positive allosteric effectors.