Kinetic and structural investigation of acyl-binding sites on avian fatty acid synthase.

Cardon, J W; Hammes, Gordon G.

doi:10.1016/s0021-9258(18)32496-7

Cited by 16 publications

(15 citation statements)

References 20 publications

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“…was found after overnight incubation of the enzyme with NBD-CoA that specifically inactivated the enzyme (Cardón & Hammes, 1983).…”

Section: Discussionmentioning

confidence: 99%

Fluorescent properties of pyrene bound at specific acylation sites of chicken liver fatty acid synthase

Anderson¹,

Hammes²

1983

Biochemistry

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The covalent modification of chicken liver fatty acid synthase by 4-(1-pyrenyl)butyryl-CoA (PBA-CoA), a fluorescent analogue of acetyl- and malonyl-CoA, has been studied. The binding isotherms and the kinetics of inactivation suggest 2 mol of PBA-CoA/mol of enzyme is specifically incorporated into the enzyme. Two classes of binding sites have been identified by determining the fluorescence lifetimes of enzyme-bound pyrene, by the quenching of enzyme-bound pyrene fluorescence with iodide, and by neutral hydroxaminolysis of both the native and denatured PBA-CoA-modified enzymes. Hydroxaminolysis of the denatured enzyme indicates that 4-(1-pyrenyl)butyric acid is esterified to both hydroxyl and thiol groups. The portion esterified to the hydroxyl is readily removed from the native enzyme by treatment with neutral hydroxylamine, indicating that the oxygen ester is unstable to hydroxylamine in the native enzyme. Iodide and acrylamide quenching of the enzyme-bound pyrene fluorescence shows that solvent access to both classes of pyrene binding sites is restricted. Iodide preferentially quenches one class of sites in the native enzyme, but these sites are not differentiated in the monomeric or denatured enzyme. The steady-state anisotropy, 0.083, indicates the enzyme-bound pyrene has considerable rotational freedom. The dynamic anisotropy can be characterized solely by a viscosity-dependent rotational correlation time of 610 ns, which is ascribed to the rotational motion of the dimeric enzyme.

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“…was found after overnight incubation of the enzyme with NBD-CoA that specifically inactivated the enzyme (Cardón & Hammes, 1983).…”

Section: Discussionmentioning

confidence: 99%

Fluorescent properties of pyrene bound at specific acylation sites of chicken liver fatty acid synthase

Anderson¹,

Hammes²

1983

Biochemistry

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“…The modification of fatty acid synthase with pyridoxal 5'phosphate has been reported (Poulose & Kolattukudy, 1980;Cardon & Hammes, 1983;Tsukamoto et al, 1983). The pyridoxal 5'-phosphate causes selective inhibition of the enoyl reductase activity in animal fatty acid synthase.…”

Section: Resultsmentioning

confidence: 99%

“…Chemicals. NADPH, NADP+, TNADP+, acetyl-CoA, malonyl-CoA, acetoacetyl-CoA, crotonyl-CoA, pyridoxal 5'phosphate, NaBH4, trifluoroacetic acid, Sephadex G-50-80, DEAE-Sephacel, D-glucose 6-phosphate, and D-glucose-6phosphate dehydrogenase (type XXIII) were from Sigma Chemical Co. TV'-Phosphopyridoxallysine was prepared as described by Cardon and Hammes (1983). Guanidine hydrochloride was from Pierce Chemical Co. L-l-(Tosylamino)-2-phenylethyl chloromethyl ketone treated trypsin was from Worthington Biochemical Corp.…”

Section: Methodsmentioning

confidence: 99%

“…Fatty acid synthase was labeled by the method of Poulose and Kolattukudy (1980), as previously described (Cardon & Hammes, 1983). To prepare pyridoxamine phosphate enzyme for proteolytic digestion, 7.2 mL of fatty acid synthase (5.0 mg/mL) in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA was mixed with 0.18 mL of 40 mM pyridoxal 5'phosphate for 15 min at 25 °C.…”

Section: Methodsmentioning

confidence: 99%

“…In this study, to characterize the NADPH-binding sites on chicken liver fatty acid synthase, the amino acid sequences associated with pyridoxal 5'-phosphate binding sites have been determined. Pyridoxal 5'-phosphate binds to a site that causes selective inhibition of the enoyl reductase activity in the enzyme and to a site that is not associated with enzymatic activity (Cardon & Hammes, 1983). In addition, the distances between the latter pyridoxal 5'-phosphate site and the two reductase sites and the distance between the two reductase sites were estimated with fluorescence resonance energy-transfer techniques.…”

mentioning

confidence: 99%

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Amino acid sequences of pyridoxal 5'-phosphate binding sites and fluorescence resonance energy transfer in chicken liver fatty acid synthase

Chang

Hammes²

1989

Biochemistry

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The amino acid sequences associated with pyridoxal 5'-phosphate binding sites in chicken liver fatty acid synthase have been determined: a site whose modification causes selective inhibition of the enoyl reductase activity and a site (site I) that is not associated with enzymatic activity. The amino acid sequences of peptides obtained by trypsin hydrolysis of the pyridoxamine 5'-phosphate labeled enzyme were determined. For the site associated with enoyl reductase activity, the sequence similarities between chicken and goose are extensive and include the sequence Ser-X-X-Lys, a characteristic structural feature of pyridoxamine enzymes. In addition, the spatial relationships between the pyridoxal 5'-phosphate binding sites and reductase site(s) have been studied with fluorescence resonance energy-transfer techniques. The distances between site I and the enoyl reductase and beta-ketoacyl reductase sites are greater than 50 and 41-44 A, respectively. The distance between the two reductase sites is greater than 49 A.

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Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

Kashem¹,

Hammes²

1988

Biochimica et Biophysica Acta (BBA) - Protein Structure and Mol

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Kinetic and structural investigation of acyl-binding sites on avian fatty acid synthase.

Cited by 16 publications

References 20 publications

Fluorescent properties of pyrene bound at specific acylation sites of chicken liver fatty acid synthase

Fluorescent properties of pyrene bound at specific acylation sites of chicken liver fatty acid synthase

Amino acid sequences of pyridoxal 5'-phosphate binding sites and fluorescence resonance energy transfer in chicken liver fatty acid synthase

Correlation of enzymatic activities and aggregation state in chicken liver fatty acid synthase

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