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.
Mukherjee
S
S
Department of Chemistry, Indian Institute of Technology Kanpur, India.
Katiyar
S S
SS
D000105:Acetyl Coenzyme A / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D010856:Columbidae; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004791:Enzyme Inhibitors / Q000494:pharmacology*; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008239:Lysine; D008316:Malonyl Coenzyme A / Q000494:pharmacology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D008316:Malonyl Coenzyme A; D011732:Pyridoxal Phosphate; D000105:Acetyl Coenzyme A; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D064429:Fatty Acid Synthases; D008239:Lysine