Interaction of formycin A-5'-triphosphate with pyruvate carboxylase

Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a competitive inhibitor of chicken liver pyruvate carboxylase with respect to ATP. The chicken liver enzyme is unable to utilise FTP as a substrate at a measureable rate, but FTP is a poor substrate for the sheep liver enzyme. When FTP binds to the enzyme, its fluorescence is enhanced and in this way the formation of enzyme-FTP complexes can be monitored. Using this property of FTP, the effect of Mg2+ and acetyl-CoA on the binding of nucleoside triphosphates to the chicken liver enzyme was examined. Mg2+ was found to enhance the binding of FTP whilst acetyl-CoA reduced the fluorescence intensity of a mixture of Mg2+, enzyme and FTP. Most probably, this was caused by a conformational change in the enzyme which changed the environment of the fluorophore.     

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker''s yeast

1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.     

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.     

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.     

Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction.

When chicken liver pyruvate carboxylase was incubated with either H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.     

Probing the allosteric activation of pyruvate carboxylase using 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate as a fluorescent mimic of the allosteric activator acetyl CoA

2′,3′-O-(2,4,6-Trinitrophenyl) adenosine 5′-triphosphate (TNP-ATP) is a fluorescent analogue of ATP. MgTNP-ATP was found to be an allosteric activator of pyruvate carboxylase that exhibits competition with acetyl CoA in activating the enzyme. There is no evidence that MgTNP-ATP binds to the MgATP substrate binding site of the enzyme. At concentrations above saturating, MgATP activates bicarbonate-dependent ATP cleavage, but inhibits the overall reaction. The fluorescence of MgTNP-ATP increases by about 2.5-fold upon binding to the enzyme and decreases on addition of saturating acetyl CoA. However, not all the MgTNP-ATP is displaced by acetyl CoA, or with a combination of saturating concentrations of MgATP and acetyl CoA. The kinetics of the binding of MgTNP-ATP to pyruvate carboxylase have been measured and shown to be triphasic, with the two fastest phases having pseudo first-order rate constants that are dependent on the concentration of MgTNP-ATP. The kinetics of displacement from the enzyme by acetyl CoA have been measured and also shown to be triphasic. A model of the binding process is proposed that links the kinetics of MgTNP-ATP binding to the allosteric activation of the enzyme.     

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.     

Improved purification and further characterization of acetyl-CoA carboxylase from cultured cells of parsley (Petroselinum hortense)

Acetyl-CoA carboxylase from irradiated cell-suspension cultures of parsley (Petroselinum hortense) has been purified to apparent homogeneity. The procedure included affinity chromatography of the enzyme on avidinmonomer--Sepharose 4B. Molecular weights of about 420000 for the native enzyme and about 220000 for the enzyme subunit were determined respectively by gel filtration or sucrose-density-gradient sedimentation and by electrophoresis in the presence of dodecyl sulfate. The purified enzyme showed an isoelectric point of 5. The enzyme carboxylated the straight-chain acyl-CoA esters of acetate, propionate, and butyrate at decreasing rates in this order. The catalytic efficiency of the carboxylase was highest when ATP existed largely as MgATP2- complex. At the optimum pH of 8 the apparent Km values for the substrates were: acetyl-CoA, 0.15 mmol/1; bicarbonate, 1 mmol/1; MgATP2-, 0.07 mmol/1. The carboxylase was inhibited by greater than 50 mmol/l NaCl, KCl, or Tris/HCl buffer. The putative allosteric activator, citrate, stimulated the enzyme only slightly at concentrations below 2 mmol/l, but strongly inhibited the carboxylase at higher concentrations. The results of these studies demonstrate that several properties of the light-inducible acetyl-CoA carboxylase of parsley cells, an enzyme of the flavonoid pathway, are remarkably similar to those of acetyl-CoA carboxylases from a variety of other organisms.     

Effect of calcium ions on pyruvate carboxylase from pigeon liver.

Pigeon liver pyruvate carboxylase (pyruvate: CO2 ligase (ADP forming), EC 6.4.1.1) shows allosteric properties similar to those of chicken or rat liver enzyme. Kinetic methods have been used to determine the effect of Ca2+ on this enzyme. The Ca2+ activation effect is absolutely dependent on the Mg2+ concentration; in the absence of Mg2+, pyruvate carboxylase has no catalytic activity. Furthermore, Ca2+ cannot replace Mg2+ and also shows a paradoxical effect on the liver enzyme activity. It is an activator at low pyruvate or Mg2+ concentrations; at increased pyruvate concentrations, however, it becomes an inhibitor. At low levels of ATP a pronounced activation of pigeon liver pyruvate carboxylase by Ca2+ has been demonstrated. The results of this communication demonstrate pigeon liver pyruvate carboxylase to be different from pyruvate carboxylase from other sources.     

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.     

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.     

Protein-serine kinase from rat epididymal adipose tissue which phosphorylates and activates acetyl-CoA carboxylase. Possible role in insulin action.

1. Most of the cyclic-nucleotide-independent acetyl-CoA carboxylase kinase activity in an extract of rat epididymal adipose tissue was evaluated from a Mono Q column by 0.175 M-NaCl at pH 7.4. The activity of the kinase in this fraction (fraction 1) was increased after exposure of intact tissue to insulin. 2. Incubation of purified adipose-tissue acetyl-CoA carboxylase with [gamma-32P]ATP and samples of fraction 1 led to the incorporation of up to 0.4 mol of 32P/mol of enzyme subunit. Most of the phosphorylation was on serine residues within a single tryptic peptide. This peptide, on the basis of two-dimensional t.l.c. analysis, h.p.l.c. and Superose 12 chromatography, appeared to be the same as the acetyl-CoA carboxylase peptide ('I'-peptide) which exhibits increased phosphorylation in insulin-treated tissue. 3. Phosphorylation of purified acetyl-CoA carboxylase by the kinase in fraction 1 was found to be associated with a parallel 4-fold increase in activity. However, increases in both phosphorylation and activity were much diminished if fraction 1 was treated by Centricon centrifugation to remove low-Mr components. Among these components was a potent inhibitor of acetyl-CoA carboxylase activity which appeared to be necessary for the kinase in fraction 1 to be fully active. 4. The inhibitor remains to be identified, but inhibition requires MgATP, although the inhibitor itself does not cause any phosphorylation of the carboxylase. No effects of insulin were observed on the activity of the inhibitor. 5. It is concluded that the kinase probably plays an important role in the mechanism whereby insulin brings about the well-established increases in phosphorylation and activation of acetyl-CoA carboxylase in adipose tissue.     

Mechanism of nicotinic acid inhibition of the reaction catalyzed by acetyl-CoA carboxylase

A kinetic analysis of the activity of acetyl-CoA carboxylase from chicken liver upon alimentary activation of lipogenesis and inhibition of this reaction by nicotinic acid was performed. It was found that the affinity of the enzyme isolated from chicken liver with stimulated lipogenesis is decreased by nicotinic acid for HCO3- but remains unchanged for ATP. The value of Vmax for ATP and the amount of the ATP used in this reaction remain unaffected. At the same time the enzyme affinity for acetyl-CoA is increased with a simultaneous decrease of Vmax. It is assumed that nicotinic acid inhibits the first step of the acetyl-CoA carboxylase-catalyzed reaction.     

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.     

Acetyl coenzyme A carboxylase. Rapid purification of the chick liver enzyme and steady state kinetic analysis of the carboxylase-catalyzed reaction

Avidin affinity chromatography was used to rapidly purify acetyl-CoA carboxylase to homogeneity in high yield from chicken liver. Dissociation of the purified carboxylase with dodecyl sulfate yielded a single size class of subunit polypeptide of 225,000 daltons. A steady state kinetic analysis of the carboxylase-catalyzed carboxylation of acetyl-CoA gave rise to intersecting line patterns in all double-reciprocal plots of initial velocity with each substrate pair, i.e. ATP . Mg and HCO3(-) and acetyl-CoA. It was concluded that the kinetic mechanism involves a quaternary complex of the enzyme, ADP, Pi, and acetyl-CoA rather than a double displacement as previously believed. The ordered addition of ATP, HCO3(-), and then acetyl-CoA, to the citrate-activated form of the carboxylase is the kinetic mechanism most consistent with the results.     

Mitochondrial location of pyruvate carboxylase in Aspergillus niger

Pyruvate carboxylase has been found in the mitochondrial fraction of two strains of Aspergillus niger along with the marker enzymes of citrate synthase and cytochrome c oxidase. The location of pyruvate carboxylase in A. nidulans was, however, confirmed to be in the cytosolic fraction. The enzyme from the former sources was dependent upon the presence of acetyl-CoA for full activity; the enzyme from A. nidulans was unaffected by the presence or absence of acetyl-CoA.     

Pyruvate carboxylase: affinity labelling of the magnesium adenosine triphosphate binding site.

The 2' , 3'-dialdehyde derivative of ATP (oATP) was prepared by periodate oxidation and on the following criteria was considered to be an effective affinity label. The magnesium complex of this derivative (Mg-oATP2) was shown to ba linear competitive inhibitor with respect to MgATP2-in both the acetyl-CoA-dependent and -independent activities of the enzyme but was a non-competitive inhibitor with respect to bicarbonate, and an uncompetitive inhibitor with respect to pyruvate. Mg-oATP was covalently bound to pyruvate carboxylase by reduction using sodium borohydride with concurrent irreversible inactivation of the enzyme...     

Regulation of kinase reactions in pig heart pyruvate dehydrogenase complex.

1. Pig heart pyruvate dehydrogenase complex is inactivated by phosphorylation (MgATP2-) of an alpha-chain of the decarboxylase component. Three serine residues may be phosphorylated, one of which (site 1) is the major inactivating site. 2. The relative rates of phosphorylation are site 1 greater than 2 greater than site 3. 3. The kinetics of the inactivating phosphorylation were investigated by measuring inactivation of the complex with MgATP2-. The apparent Km for the Mg complex of ATP was 25.5 microM; ADP was a competitive inhibitor (Ki 69.8 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 2.8 microM). Inactivation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA. 4. The kinetics of additional phosphorylations (predominantly site 2 under these conditions) were investigated by measurement of 32P incorporation into non-radioactive pyruvate dehydrogenase phosphate containing 3-6% of active complex, and assumed from parrallel experiments with 32P labelling to contain 91% of protein-bound phosphate in site 1 and 9% in site 2. 5. The apparent Km for the Mg complex of ATP was 10.1 microM; ADP was a competitive inhibitor (Ki 31.5 microM) and sodium pyruvate an uncompetitive inhibitor (Ki 1.1 mM). 6. Incorporation was accelerated by increasing concentration ratios of NADH/NAD+ and of acetyl-CoA/CoA, although it was less marked at the highest ratios.     

The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxaloacetate

Fatty acid synthesis via the citrate cleavage pathway requires the continual replenishment of oxaloacetate within the mitochondria, probably by carboxylation of pyruvate. Malic enzyme, although present in adipose tissue, is completely localized in the cytoplasm and has insufficient activity to support lipogenesis. Pyruvate carboxylase was found to be active in both the mitochondria and cytoplasm of epididymal adipose tissue cells; it was dependent on both ATP and biotin. Alteractions in dietary conditions induced no significant changes in mitochondrial pyruvate carboxylase activity, but the soluble activity was depressed in fat-fed animals. The possible importance of the soluble activity in lipogenesis lies in its participation in a soluble malate transhydrogenation cycle with NAD malate dehydrogenase and malic enzyme, whereby a continual supply of NADPH is produced. Consequently, the pyruvate carboxylase in adipose tissue both generates mitochondrial oxaloacetate for the citrate cleavage pathway and supplies soluble NADPH for the conversion of acetyl-CoA to fatty acid.