Fluorescent properties of pyrene bound at specific acylation sites of chicken liver fatty acid synthase.
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.
D000214:Acyl Coenzyme A / Q000494:pharmacology*; D000215:Acylation; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D064429:Fatty Acid Synthases / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011485:Protein Binding; D011721:Pyrenes / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
D000214:Acyl Coenzyme A; D005456:Fluorescent Dyes; D011721:Pyrenes; C035257:1-pyrenebutyryl-coenzyme A; D064429:Fatty Acid Synthases