Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Katiyar, Sarvagya S.; Briedis, Anita V.; Porter, John W.

doi:10.1016/0003-9861(74)90199-4

Cited by 20 publications

(7 citation statements)

References 14 publications

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“…USA 80 (1983) [3] [5] with KmM 2.0 MM (pH 7.0), but with kcat only 7% of the value observed for the "normal" reaction. A similar observation has been reported for pigeon liver fatty acid synthase (3)(4)(5) and was attributed to decarboxylation of the malonyl moiety bound covalently to 4'-phosphopantatheine, to yield the corresponding acetyl derivative. Purification of the Mal-CoA by high-performance liquid chromatography on a C18 column eluted with acetonitrile/10 mM ammonium acetate, pH 4.5, 9:91 (vol/vol), reduced kcat to 0.2% of the value observed for the normal reaction.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Steady-state kinetic study of fatty acid synthase from chicken liver.

Cox¹,

Hammes²

1983

Proc. Natl. Acad. Sci. U.S.A.

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The steady-state kinetics of chicken liver fatty acid synthase has been studied over the pH range 5.9-8.6 The fatty acid synthase from chicken liver is a multienzyme complex that catalyzes the synthesis of palmitic acid from acetyl-CoA (Ac-CoA), malonyl-CoA (Mal-CoA), and NADPH. The synthesis is initiated by the acetyl group and involves the condensation of seven malonyl groups on to the growing chain. Each condensation reaction is followed by reduction to an alcohol, dehydration, and reduction of the resultant carbon-carbon double bond; both reduction steps utilize NADPH. The overall reaction is Ac-CoA + 7 Mal-CoA + 14 NADPH + 14H+ [1] palmitic acid + 8 CoA + 7 C02 + 6 H20 + 14 NADP+.Steady-state kinetic studies of the overall reaction and the partial reactions of the pigeon liver enzyme have been carried out (1-5). The results are consistent with the mechanism involving three successive covalently bound intermediates, but quantitative estimates of the kinetic parameters are in general not available. This is primarily due to the restricted range of substrate concentrations employed. In this work, the steadystate kinetics of the chicken liver fatty acid synthase was studied over a wide range of substrate concentrations and pH. This has permitted quantitative determination of the steady-state Fatty Acid Synthase Preparation. Fatty acid synthase from chicken liver was prepared and assayed as described (6). The specific activity of the enzyme was ""1.6 ,tmol of NADPH per min/mg at 250C. Protein concentrations were determined by using an absorption coefficient for fatty acid synthase of 0.965 cm2/mg at 279 nm and by assuming Mr = 500,000 (7). Enzyme concentrations were normally ---2 x 10-8 M, but separate experiments showed the observed rates to be linear in enzyme concentration over the range 2 X 10-9 to 5 X 10-8 M; above these concentrations, the rates were too fast to measure by conventional methods. The concentrations of the three substrates were varied over a wide range as follows: Ac-CoA, 0.6-120 ,uM; Mal-CoA, 1.3-160 kLM; NADPH, 1.5-150 ,uM. Concentrations of Ac-CoA and Mal-CoA were determined spectrophotometrically by using an extinction coefficient of 1.5 X I04 M-1 cm-' at 260 nm (5, 9). Stock solutions of the substrates in pure water were stored at 0°C and those of the enzyme in 0.1 M potassium phosphate, pH 7.0/1 mM EDTA at room temperature. Under these conditions the decrease in activity of the solutions during the time required for a typical set of measurements (2-4 hr) was <5%.Reactions were initiated by adding 50 ,1l of the stock enzyme solution to an appropriate solution of the substrates (1.3 ml) in a 1-cm path length spectrophotometric cell thermostated at 250C. Different enzyme solutions (all from a single preparation) were standardized spectrophotometrically: measurement of the initial velocities under standard conditions, 0.1 M potassium phosphate, pH 7.0/1 mM EDTA/100 ,uM Mal-CoA/50 p.M Ac-CoA/100 p.M NADPH, showed that the specific activities were constant to within ±10%. At pH '6, ...

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Section: Resultsmentioning

confidence: 99%

“…Steady-state kinetic studies of the overall reaction and the partial reactions of the pigeon liver enzyme have been carried out (1)(2)(3)(4)(5). The results are consistent with the mechanism involving three successive covalently bound intermediates, but quantitative estimates of the kinetic parameters are in general not available.…”

mentioning

confidence: 99%

Steady-state kinetic study of fatty acid synthase from chicken liver.

Cox¹,

Hammes²

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…A modification of the assay described by Katiyar et al [14] was used to measure the inactivation of the β-ketoacyl synthase reaction catalysed by FAS. This partial reaction monitors the formation of triacetic acid lactone at 283 nm from acetyl-CoA and malonyl-CoA in the absence of NADPH [15].…”

Section: Inhibition Of Fas β-Ketoacyl Synthase Activitymentioning

confidence: 99%

Characterization of the inactivation of rat fatty acid synthase by C75: inhibition of partial reactions and protection by substrates

Rendina

Cheng

2005

Biochemical Journal

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C75, a synthetic inhibitor of FAS (fatty acid synthase), has both anti-tumour and anti-obesity properties. In this study we provide a detailed kinetic characterization of the mechanism of in vitro inhibition of rat liver FAS. At room temperature, C75 is a competitive irreversible inhibitor of the overall reaction with regard to all three substrates, i.e. acetyl-CoA, malonyl-CoA and NADPH, exhibiting pseudo-first-order kinetics of the complexing type, i.e. a weak non-covalent enzyme-inhibitor complex is formed before irreversible enzyme modification. C75 is a relatively inefficient inactivator of FAS, with a maximal rate of inactivation of 1 min(-1) and an extrapolated K(I) (dissociation constant for the initial complex) of approx. 16 mM. The apparent second-order rate constants calculated from these values are 0.06 mM(-1).min(-1) at room temperature and 0.21 mM(-1).min(-1) at 37 degrees C. We also provide experimental evidence that C75 inactivates the beta-ketoacyl synthase (3-oxoacyl synthase) partial activity of FAS. Unexpectedly, C75 also inactivates the enoyl reductase and thioesterase partial activities of FAS with about the same rates as for inactivation of the beta-ketoacyl synthase. In contrast with the overall reaction, the beta-ketoacyl synthase activity and the enoyl reductase activity, substrates do not protect the thioesterase activity of rat liver FAS from inactivation by C75. These results differentiate inactivation by C75 from that by cerulenin, which only inactivates the beta-ketoacyl synthase activity of FAS, by forming an adduct with an active-site cysteine. Interference by dithiothreitol and protection by the substrates, acetyl-CoA, malonyl-CoA and NADPH, further distinguish the mechanism of C75-mediated inactivation from that of cerulenin. The most likely explanation for the multiple effects observed with C75 on rat liver FAS and its partial reactions is that there are multiple sites of interaction between C75 and FAS.

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“…An alternate explanation fora lack of acetyl-CoA dependency could be that the synthetase itself generates the acetyl moiety from malonyl-CoA. The purified fatty acid synthetase from pigeon liver has been shown to catalyze the synthesis of fatty acids from only malonyl-CoA and NADPH (Katiyar et al, 1974). The experimental results indicated that the malonyl moiety was decarboxylated when covalently linked to 4'-phosphopantetheine of the enzyme to give the bound acetyl moiety.…”

Section: Properties Of Fatty Acid Synthetasementioning

confidence: 99%

One-step purification and properties of a two-peptide fatty acid synthetase from the uropygial gland of the goose

Buckner¹,

Kolattukudy²

1976

Biochemistry

117

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Cell-free extracts from the uropygial gland of goose catalyzed the incorporation of malonyl-CoA into normal fatty acids and methylmalonyl-CoA into multimethyl branched acids with NADPH as the preferred reductant (J. S. Buckner and P.E. Kolattukudy (1975), Biochemistry 14, 1771). Purification of fatty acid synthetase from this extract was accomplished in one step by gel filtration with Sepharose 4B. Homogeneity of the fatty acid synthetase was shown by analytical ultracentrifugation, immunodiffusion assays, polyacrylamide disc gel electrophoresis, and sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. At a pH of 7.0, apparent Km values of 3.6 X 10(-5) M and 1.5 X 10(-5) M were calculated for malonyl-C0A and NADPH, respectively. The major products synthesized by the enzyme from malonyl-CoA and methylmalonyl-C0A were free hexadecanoic acid and free 2, 4, 6, 8-tetramethyldecanoic acid, respectively, with acetyl-CoA as primer. A molecular weight value of 547 000 was determined for the goose fatty acid synthetase by sedimentation equilibrium centrifugation. The purified enzyme had an s20,w of 13.5S and was partially dissociated in low-ionic strength buffer into a 9.3S species, and this dissociation was accompanied by a corresponding partial inactivation of the enzymatic activity. Reassociation and reactivation of the partially dissociated fatty acid synthetase were accomplished in either 0.2 M KCl or 200 muM NADPH. These properties of the goose enzyme are similar to those of other animal fatty acid synthetases, as was the amino acid composition. Dissociation of the purified enzyme with sodium dodecyl sulfate resulted in only two equal molecular weight polypeptides (269 000), as determined by sodium dodecyl sulfate polyacrylamide disc gel electrophoresis. Injection of labeled pantothenic acid into the uropygial gland resulted in the synthesis of labeled fatty acid synthetase in which the label appeared to be located exclusively in the 4'-phosphopantotheine moiety. Analysis of the labeled enzyme by gel filtration and polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate showed that the labeled pantothenate was contained exclusively in the half molecular weight moiety. The enzyme contained one 4'-phosphopantetheine residue per subunit (269 000), as determined by measurement of the taurine generated by hydrolysis of performic acid-treated enzyme. Sodium dodecyl sulfate-activated proteolytic activity was shown to be associated with goose fatty acid synthetase, and this proteolysis was shown to result in the formation of small-molecular-weight protein fragments (less than 200 000) during treatment of the enzyme with sodium dodecyl sulfate. This proteolysis could be prevented by diisopropyl fluorophosphate and p-chloromercuribenzoate. These results strongly suggest that the goose uropygial gland fatty acid synthetase consists of two multifunctional polypeptide subunits, each containing one covalently linked 4'-phosphopantetheine.

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Synthesis of fatty acids from malonyl-CoA and NADPH by pigeon liver fatty acid synthetase

Cited by 20 publications

References 14 publications

Steady-state kinetic study of fatty acid synthase from chicken liver.

Steady-state kinetic study of fatty acid synthase from chicken liver.

Characterization of the inactivation of rat fatty acid synthase by C75: inhibition of partial reactions and protection by substrates

One-step purification and properties of a two-peptide fatty acid synthetase from the uropygial gland of the goose

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