Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate.
Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D008239:Lysine; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D011732:Pyridoxal Phosphate; D000429:Alcohol Oxidoreductases; C021461:beta-ketoacyl thioester reductase; D064429:Fatty Acid Synthases; D008239:Lysine