Pigeon Liver Fatty Acid Synthetase Research Articles

Inactivation of 3-oxoacyl synthetase activity of pigeon liver fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-coenzyme A.

Pigeon liver fatty acid synthetase was inactivated by treatment with S-(4-bromo-2,3-dioxobutyl)-CoA. This inactivation is fast, irreversible and dependent on both time and inhibitor concentration. It is also specific and selective as only the 3-oxoacyl synthetase and acetyl-CoA transacylase component activities, out of the seven component activities possessed by the fatty acid synthetase complex, were inhibited. The binding of bromo-dioxobutyl-CoA to the enzyme is covalent as evidenced by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The modified enzyme showed characteristic absorption peaks at 266 nm and 315 nm that are identical to the absorption maxima of pure inhibitor. All the available evidence suggests that the mechanism of inhibition of fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-CoA involved its binding to critical thiols, namely the cysteine — SH of the condensing enzyme and cysteamine — SH of the 4′-phosphopantetheine prosthetic group. Both subsrates, acetyl-CoA and malonyl-CoA, also protected the enzyme against inhibition by S-(4-bromo-2,3-dioxobutyl)-CoA. Comparison of high-voltage electrophoretic patterns of peptic peptides obtained from [1-14C]acetyl-labeled native and S-(4-bromo-2,3-dioxobutyl)-CoA-inactivated fatty acid synthetase confirmed the involvement of both cysteine and 4′-phosphopantetheine — SH groups in the binding with S-(4-bromo-2,3-dioxobutyl)-CoA. Scatchard analysis of the results of binding studies has established that 4 mol of the inhibitor are bound/mol of the enzyme complex, which indicates the presence of two moles condensing activities and two moles 4′-phosphopantetheine prosthetic groups/mole of enzyme. Our data do not support a half-site reactivity for the fatty acid synthetase as proposed earlier [Clements et al. (1979) Biochem. Biophys. Res. Commun. 86, 278–284].

Inhibition of Pigeon Liver Fatty Acid Synthetase by Specific Modification of Lysine Residues with 2, 4, 6–Trinitrobenzenesulphonic Acid

Pigeon liver fatty acid synthetase was inactivated irreversibly by 2,4,6-trinitrobenzenesulphonic acid (TNBS). Biphasic inactivation of the enzyme was observed with the inhibitor. NADPH provided protection to the enzyme against inactivation by TNBS and the extent of protection increased with NADPH concentration indicating that the essential lysine residues are present at the NADPH binding site. The stoichiometric results with TNBS showed that 4 mol of lysine residues are modified per mole of fatty acid synthetase upon complete inactivation. The rapid reaction of two amino groups per enzyme molecule led to the loss of 60% of the enzyme activity. These approaches suggested that two lysine residues present at the active site are essential for the enzymatic activity of fatty acid synthetase.

Inactivation of enoyl-CoA reductase in pigeon liver fatty acid synthetase by pyridoxal 5'-phosphate: evidence for the presence of one lysine residue at the active site.

Pigeon liver fatty acid synthetase (FAS) was rapidly inactivated by pyridoxal 5'-phosphate (PLP). Assays of the partial activities of the PLP-treated synthetase showed that only the enoyl-CoA reductase was decreased significantly. The inactivation of both the overall activity and enoyl-CoA reductase activity of FAS by PLP could be reversed by dialysis or dilution but not by reduction with sodium borohydride. Malonyl-CoA and acetyl-CoA did not protect the enzyme, whereas NADPH provided 68% protection against PLP-inactivation indicating that PLP modified lysine residues present at or near the co-enzyme binding site. PLP-treated enzyme after reduction with sodium borohydride exhibited fluorescence with a maximum at 397 nm (irradiation at 325 nm). Stoichiometric analysis showed that modification of four lysine residues per enzyme molecule resulted in complete inactivation of the overall and enoyl-CoA reductase activities of FAS. NADPH prevented the inactivation by protecting two of these lysine residues from modification, suggesting the presence of two essential lysine residues per enzyme molecule. These results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.

Evidence for the essential histidine at the NADPH binding site of enoyl-CoA reductase domain of pigeon liver fatty acid synthetase.

Pigeon liver fatty acid synthetase was inactivated by stoichiometric concentrations of diethylpyrocarbonate (DEP). The second order rate constant for the loss of synthetase activity was similar to the value for enoyl-CoA reductase indicating that ethoxyformylation destroys the ability of the enzyme to reduce the unsaturated acyl intermediate, without significant effect on beta-ketoacyl reductase activity. NADPH provided protection to the enzyme against inactivation by DEP indicating that essential histidine residues are present at the active site. DEP-modified enzyme showed a characteristic absorption maxima at 240 nm confirming the formation of ethoxyformic histidine. The reversal of inactivation by hydroxylamine and a pKa value of 7.0 obtained from the pH-rate profile for inactivation again confirmed the specificity of DEP for histidine. Stoichiometric results showed that two moles of histidine residue per mole of enzyme are essential for the activity of FAS.

Purification of nucleotide-requiring enzymes by immunoaffinity chromatography.

Monospecific (affinity-purified) anti-(yeast glucose-6-phosphate dehydrogenase) IgG inhibits three different NADPH-requiring enzymes, chicken liver dihydrofolate reductase, pigeon liver fatty acid synthetase and chicken liver malic enzyme. The inhibition of all three enzymes was approx. 50% in a 2h incubation with 100 micrograms of IgG. Similarly, with several different NADH-requiring enzymes, an immunocrossreactivity was observed. Monospecific anti-(rabbit muscle glyceraldehyde-3-phosphate dehydrogenase) IgG inhibited yeast alcohol dehydrogenase and pig heart malate dehydrogenase by 39% and 55% respectively. The cross-reactivity observed was tested by affinity chromatography. Immunoaffinity columns made with each monospecific IgG were able to bind each of the enzymes it immunotitrated. Enzymes were eluted with a nondenaturing solvent with little loss of activity. The immunoaffinity column with monospecific anti-(glucose-6-phosphate dehydrogenase) IgG as the bound ligand was also used to purify partially (over 150-fold) both isocitrate dehydrogenase and dihydrofolate reductase from crude rat liver homogenate.

Fatty acid synthetase, malic enzyme and other NADP + binding dehydrogenases have similar antigenic determinant(s) at the NADPH binding domain

Rabbit IgG prepared against malic enzyme inhibits pigeon liver fatty acid synthetase activity. By screening a number of polyclonal antibodies raised to several dehydrogenases, we have discovered that there is a direct relationship between this cross-reactivity and the existence of a common structural domain whose function is the binding of NADP + NADPH . The presence of NADP + NADPH in small concentrations protects against this immunoinhibition, thereby indicating a competitive binding of nucleotide and site-specific antibody at the antigenic domain. These data provide further support for the existence of an antigenic domain in the NADPH binding region of dehydrogenases which is responsible for the production of NADPH site-specific antibodies in polyclonal antisera.

Degradation of pigeon liver fatty acid synthetase in the absence of exogenous proteinases

The homogeneity of pigeon liver fatty acid synthetase has been rigorously tested by physicochemical techniques and crossed-rocket immunoelectrophoresis. The enzyme has also been incubated for 1 h at 100°C in 2% sodium dodecyl sulfate and 0.1 M dithiothreitol. The number of protein components on gel electrophoresis and of dansylated amino acids increased as a function of incubation time. Furthermore, the minor proteins observed after gel electrophoresis cross-reacted with antibody raised to the synthetase. Proteolysis was not chemically mediated by the detergent, the reducing agent or the buffer conditions chosen. Several commercially prepared proteins were not degraded by this procedure, and two proteins were recalcitrant to hydrolysis when included in the same incubation mixture as the synthetase. The inclusion of certain microbial proteinase inhibitors decreased the amount of degradation. This demonstrated that hydrolysis of the synthetase is mediated by a specific vertebrate enzyme which retains activity under denaturing conditions at 100°C. Further degradation is also observed after individual treatment of four limited digestion products from the pigeon liver fatty acid synthetase, suggesting the possibility of an inherent proteolytic activity within the complex.

Liberation, purification, and properties of thioesterase component of pigeon liver fatty acid synthetase complex

Proteolysis of pigeon liver fatty acid synthetase with elastase results in the quantitative cleavage of the thioesterase component from the enzyme complex. This thioesterase component is two or three times more active catalytically in the isolated state than in the native fatty acid synthetase, and its activity is not affected by the presence or absence of reducing thiols. The proteolytically cleaved thioesterase is separated from the core enzyme in one step by size-exclusion chromatography on a Sephadex G-75 column. The peptide obtained by gel permeation is homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. Size-exclusion chromatography on Bio-Gel A 0.5 m and Sephadex G-75 columns, sucrose density gradient ultracentrifugation, and N- terminal amino acid analysis also indicate that the proteolytically cleaved thioesterase is homogeneous. The sedimentation coefficient of the thioesterase is approximately 2.9 S. Proteolytic cleavage with elastase also quantitatively releases the [1,3- 14C]- or [1,3- 3H] diisopropylphosphofluoridate-labeled thioesterase component from the correspondingly labeled fatty acid synthetase. Binding studies with 14C- or 3H-labelled diisopropylphosphofluoridate and fatty acid synthetase show that 2 mol of the label are bound per mol of the enzyme when complete loss of fatty acid-synthesizing activity occurs. The molecular weight of the thioesterase component is estimated to be 36000 by size-exclusion chromatography, SDS-polyacrylamide gel electrophoresis and amino acid analysis.

Isolation and characterization of an acyl carrier protein from pigeon liver fatty acid synthetase by controlled proteolysis with elastase

Controlled proteolytic cleavage of 4′-phospho[ 14C]pantetheine-labeled pigeon liver fatty acid synthetase generates two 4′-phospho[ 14C]pantetheine-labeled peptides, E c1 and E c2. These are separated from each other and the core enzyme by gel permeation chromatography on a Sephadex G-75 column. The two radioactively labeled peptides constitute 50% of the radioactivity initially present in the 4′-phospho[ 14C]pantetheine-labeled fatty acid synthetase. The remaining label in the core enzyme is released quantitatively by proteolytic cleavage with trypsin. The molecular weights of E c1 and E c2 peptides, as determined by size exclusion chromatography and SDS-polyacrylamide gel electrophoresis, are 12000 and 6000, respectively. Both the higher and lower molecular weight peptides are homogeneous with respect to size and charge, as shown by polyacrylamide gel electrophoresis in the presence and absence of SDS. The higher molecular weight peptide, E c1, is characterized as an acyl carrier protein by the transacylation reaction between the unlabeled E c1 peptide and radioactively labeled acetyl- and malonyl-CoA. Since E c2 peptide also contains the prosthetic group present in the E c1 peptide, the E c2 peptide appears to result from the proteolytic cleavage of the higher molecular weight peptide, E c1. Amino acid composition of the acyi carrier protein shows the presence of 1 mol of 4′-phosphopantetheine per mol of protein. 2 mol of acyl carrier protein are present per mol of the fatty acid synthetase. The amino acid analysis is in good agreement with the molecular weight of the E c1 peptide, as determined by gel filtration and SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid analysis of this peptide shows the presence of an arginine residue.

Isolation and partial purification of elastase-released peptide domains that contain the partial activities of pigeon liver fatty acid synthetase

Proteolytic cleavage of pigeon liver fatty acid synthetase with elastase (4% w/w) for 5 h yields two peptides, designated II and IV, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of peptides II and IV are 130,000 and 86,000, respectively. These peptides are separated by dye-ligand chromatography on an Affi-Gel Blue column. Peptide II is found to contain the domains of β-ketoacyl and enoyl reductases and β-hydroxyacyl dehydrase, whereas peptide IV contains the domains of acetyl- and malonyl-CoA:pantetheine transacylases.

The involvement of a lysine residue at the active site of the enoyl reductase of pigeon liver fatty acid synthetase

The reaction of pyridoxal-5′-phosphate with lysine residues of pigeon and rat liver fatty acid synthetases has been investigated. The enzyme is reversibly inactivated by this compound inasmuch as overall and enoyl reductase activities are lost as a function of time. β-Ketoacyl reductase activity and other component activities of the enzyme are not affected. Spectrophotometric assays, as well as amino acid analysis of the acid hydrolysate of NaB 3 H 4 -reduced enzyme-pyridoxal phosphate complex, have established that the inactivation of the enzyme is due to Schiff base formation with e-amino groups of lysine residues that are essential for the enoyl reductase component activity of fatty acid synthetases.

Limited elastase digestion of pigeon liver fatty acid synthetase with retention of all partial enzyme activities.

Pigeon liver fatty acid synthetase which contains two subunits of 240,000 daltons each has been treated with elastase. This treatment yields four protein fragments which can be separated on sodium dodecyl sulfate (SDS)-gel electrophoresis. After the subunit protein has been treated with elastase, all of the partial enzyme activities catalyzed by the complex are present, but enzyme activity for fatty acid synthesis is lost. The formation of protein fragments during proteolysis has been followed by densitometric scanning of the SDS gels. The results of these scans have suggested that (a) there are two peptide components present in the highest molecular weight band, (b) both are rapidly digested to yield the second and third largest peptides, and (c) a further cleavage of the third largest peptide gives rise to the smallest of the four major peptides. Crossed-rocket immunoelectrophoretic analysis of the four protein fragments has confirmed these conclusions and established also that the three smallest peptides are homogeneous. Each of the four peptides has been isolated by preparative SDS-gel electrophoresis, and antibody to one has been prepared. This antibody fraction immunotitrates overall fatty acid synthetase activity and immunoprecipitates the native enzyme. Immunoelectrophoresis of the four elastase-digested synthetase products against this antibody showed some cross-reactivity with a peptide that was neither the precursor nor the product of the immunogen. This cross-reacting antibody was removed by reaction with the nonrelated protein to yield antibody specific for one region of the fatty acid synthetase complex.

Role of cysteine and 4′-phosphopantetheine in the inactivation of pigeon liver fatty acid synthetase by S-(4-bromo-2,3-dioxobutyl)-coenzyme a

S-(4-bromo-2,3-dioxobutyl)-CoA has been used as an inhibitor of fatty acid synthetase from pigeon liver. This affinity label selectively and irreversibly inhibits the acetyl transacylase and β-ketoacyl synthetase reactions of this multienzyme complex. Binding studies with [ 3H]-labeled bromodioxobutyl-CoA have established that four mol of the inhibitor are bound per mol of the enzyme complex, and that the radioactivity of this compound is covalently bound to cysteine and 4′-phosphopantetheine moieties. Other partial reactions of fatty acid synthesis are unaffected by bromodioxobutyl-CoA.

Isolation of thioesterase and acyl carrier protein activities liberated by elastase digestion of pigeon liver fatty acid synthetase

Proteolysis of pigeon liver fatty acid synthetase with elastase cleaves the thioesterase component and an acyl carrier protein-containing peptide from the multienzyme complex. These proteins are then separated in one step by gel filtration on a Sephadex G-75 column. Each of the eluted proteins is homogeneous, as determined by polyacrylamide gel electrophoresis. The molecular weight of each has been estimated to be 36,000 and 12,000 daltons, respectively.

Affinity purification of anti-pigeon liver fatty acid synthetase immunoglobulin and comparative immunoreactivity of the catalytic reactions

Rabbit anti-pigeon liver fatty acid synthetase antibody was prepared by affinity chromatography on Sepharose-fatty acid synthetase to near monospecificity (98% or more) as shown by immunodiffusion plates and rocket immunoelectrophoresis. Immunotitrations of the highly purified monospecific antibody against the overall activity and partial activities of fatty acid synthetase were then carried out. Only 6 mol of antibody/mol of enzyme was required to inactivate overall fatty acid synthetase activity and the condensation reaction, while 12 to 18 mol were required to partially inactivate the β-ketoacyl reductase and the malonyl- and acetyl-CoA transferases. Palmitoyl-CoA thioesterase (deacylase) activity was not inhibited by the antibody. The degree of inactivation of the partial reactions by antibody was not affected by dissociation of the fatty acid synthetase. Immunoprecipitation of the enzyme indicated that there are approximately 35 immunoreactive sites on the fatty acid synthetase molecule. The possible implications of these results to an understanding of the structural organization of pigeon liver fatty acid synthetase and its antigenic determinants are discussed.

Subunits of fatty acid synthetase complexes: Comparative study of enzyme activities and properties of the half-molecular weight nonidentical subunits of fatty acid synthetase complexes obtained from rat, human, and chicken liver and yeast

Rat, human, and chicken liver and yeast fatty acid synthetase complexes were dissociated into half-molecular weight nonidentical subunits of molecular weight 225,000–250,000 under the same conditions as used previously for the pigeon liver fatty acid synthetase complex [Lornitzo, F. A., Qureshi, A. A., and Porter, J. W. (1975) J. Biol. Chem. 250, 4520–4529]. The separation of the half-molecular weight nonidentical subunits I and II of each fatty acid synthetase was then achieved by affinity chromatography on Sepharose ϵ-aminocaproyl pantetheine. The separations required, as with the pigeon liver fatty acid synthetase, a careful control of temperature, ionic strength, pH, and column flow rate for success, along with the freezing of the enzyme at −20 °C prior to the dissociation of the complex and the loading of the subunits onto the column. The separated subunit I (reductase) from each fatty acid synthetase contained β-ketoacyl and crotonyl thioester reductases. Subunit II (transacylase) contained acetyl- and malonyl-coenzyme A: pantetheine transacylases. Each subunit of each complex also contained activities for the partial reactions, β-hydroxyacyl thioester dehydrase (crotonase), and palmitoyl-CoA deacylase. The specific activities of a given partial reaction did not vary in most cases more than twofold from one fatty acid synthetase species to another. The rat and human liver fatty acid synthetases required a much higher ionic strength for stability of their complexes and for the reconstitution of their overall synthetase activity from subunits I and II than did the pigeon liver enzyme. On reconstitution by dialysis in high ionic strength potassium phosphate buffer of subunits I and II of each complex, 65–85% of the control fatty acid synthetase activity was recovered. The rat and human liver fatty acid synthetases cross-reacted on immunoprecipitation with antisera. Similarly, chicken and pigeon liver fatty acid synthetases crossreacted with their antisera. There was, however, no cross-reaction between the mammalian and avian liver fatty acid synthetases and the yeast fatty acid synthetase did not cross-react with any of the liver fatty acid synthetase antisera.

Mechanisms and regulation of biosynthesis of saturated fatty acids.

Mechanisms and regulation of biosynthesis of saturated fatty acids.

Cell-free synthesis of pigeon liver fatty acid synthetase

Pigeon liver fatty acid synthetase proteins (apo- and holo-forms) have been synthesized in a cell-free system reconstituted from polysomes and a soluble enzyme fraction. Identification of the cell-free synthesized products as fatty acid synthetase was achieved by affinity chromatography, by immuno-precipitation and by the simultaneous conversion of both the authentic carrier protein and the in vitro synthesized products from the holo- to the apo-form of the synthetase. The reverse conversion was also effected.

Separation of two active forms (holo- a and holo- b) of pigeon liver fatty acid synthetase and their interconversion by phosphorylation and dephosphorylation

Apo-, holo-a- and holo-b-pigeon liver fatty acid synthetases4 were separated by affinity gel chromatography under conditions identical to those used for the separation of apo and holo forms [Qureshi etal. (1975) Biochem. Biophys. Res. Commun. 64, 836-844], except that an additional elution step at a high salt concentration, pH 7.0, and room temperature was included. The interconversion of the two forms of holo-fatty acid synthetase was then carried out invitro. In the presence of Mg++ and a phosphatase preparation, holo-b was converted to holo-a. The reverse conversion, holo-a to holo-b, was carried out in the presence of ATP and a kinase fraction prepared from the 100, 000 g supernatant of liver homogenate. These results indicate that the interconversion of holo-a- and holo-b-fatty acid synthetases occurs by phosphorylation-dephosphorylation. These results also suggest the possibility that this process may be a mechanism of short term regulation of liver fatty acid synthetase activity.

Functional deacylases of pigeon liver fatty acid synthetase complex.

Fatty acid synthetase complex (Mr = 500,000) purified from pigeon liver homogenates is inactivated by phenylmethylsulfonyl fluoride. A well characterized inhibitor of serine esterases. Pseudounimolecular kinetics are followed at all inhibitor concentrations studied (0.05 to 1.0 mM). The second order rate constant obtained at pH 7.0, 30 degrees in 0.05 M potassium phosphate, 1 mM EDTA is 250 plus or minus 10 M-1 min-1 and appears to be independent of pH between 6 and 7.9. The inactivation of the enzyme complex appears to be selective since only one of the several component enzymes of fatty acid synthesis, palmityl-CoA deacylase, is inhibited. Acetyl- and malonyl-CoA-pantetheine transacylase activities as well as the kinetics of the reduction and dehydration steps are nearly identical for the native and the modified enzymes. The rate of approach of the condensation-CO2 exchange reaction (substrates: hexanoyl-CoA, malonyl-CoA, CoA, and H14CO3-) is slightly slower in the modified enzyme, though this change is not large enough to account for total loss of activity for fatty acid synthesis. The rate of loss of palmityl-CoA deacylase activity at a constant inhibitor concentration follows biphasic kinetics. Complete inactivation is achieved only after 2 mol of the inhibitor are bound per mol of the enzyme complex. Acetyl-, butyryl-, and hexanoyl-CoA thioesters (at 1.0 mM concentrations) protect the enzyme complex against inactivation by phenylmethylsulfonyl fluoride whereas CoA has no effect. Malonyl-CoA on the other hand, promotes inhibitor-mediated inactivation. Of the N-acetyl cysteamine derivatives tested, S-acetyl-N-acetyl cysteamine (at 10 mM) gives almost complete protection against inactivation whereas S-acetoacetyl-, S-beta-hydroxybutyryl-, and S-crotonyl-N-acetyl cysteamine thioesters exhibit either slight or no protection. These data demonstrate that phenylmethylsulfonyl fluoride is a selective reagent for the inactivation of functional fatty acyl deacylase component(s) of the pigeon liver fatty acid synthetase complex, and that it has no effect on malonyl or acetyl transacylases. The data are also in accord with the postulation that the inhibitor interacts at two catalytic centers of the enzyme complex. Furthermore, the patterns of protective effects shown by saturated acyl-CoA asters and malonyl-CoA point to different mechanisms of deacylation for these esters.