The presence of a histidine residue at or near the NADPH binding site of enoyl reductase domain on the multifunctional fatty acid synthetase of chicken liver.
Chemical modification of chicken liver fatty acid synthetase with the reagent ethoxyformic anhydride causes inactivation of the palmitate synthetase and enoyl reductase activities of the enzyme complex, but without significant effect on its beta-ketoacyl reductase or beta-ketoacyl dehydratase activity. The second-order rate constant of 0.2 mM-1 X s-1 for loss of synthetase activity is equal to the value for enoyl reductase, indicating that ethoxyformylation destroys the ability of the enzyme to reduce the unsaturated acyl intermediate. The specificity of this reagent for histidine residues is indicated by the appearance of a 240 nm absorption band for ethoxyformic histidine corresponding to the modification of 2.1 residues per enzyme dimer, and by the observation that the modified enzyme is readily reactivated by hydroxylamine. A pK value of 7.1 obtained by studies of the pH rate-profile of inactivation is consistent with that of histidine. Moreover, inactivation by ethoxyformic anhydride is unaffected by reversely blocking essential SH groups of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid), and therefore does not involve the reaction of these groups. The reaction of tyrosyl groups is excluded by an unchanged absorption at 278 nm. In other experiments, it was shown that inactivation of synthetase is protected by pyridine nucleotide cofactors and nucleotide analogs containing a 2'-phosphate group, and is accompanied by the loss of 2.4 NADPH binding sites. These results implicate the presence of a histidine residue at or near the binding site for 2'-phosphate group of pyridine nucleotide in the enoyl reductase domain of the synthetase.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002645:Chickens; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000037:antagonists & inhibitors
D000345:Affinity Labels; D006639:Histidine; D009249:NADP; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D064429:Fatty Acid Synthases; D004047:Diethyl Pyrocarbonate