Evidence for the essential histidine at the NADPH binding site of enoyl-CoA reductase domain of pigeon liver fatty acid synthetase.
Pigeon liver fatty acid synthetase was inactivated by stoichiometric concentrations of diethylpyrocarbonate (DEP). The second order rate constant for the loss of synthetase activity was similar to the value for enoyl-CoA reductase indicating that ethoxyformylation destroys the ability of the enzyme to reduce the unsaturated acyl intermediate, without significant effect on beta-ketoacyl reductase activity. NADPH provided protection to the enzyme against inactivation by DEP indicating that essential histidine residues are present at the active site. DEP-modified enzyme showed a characteristic absorption maxima at 240 nm confirming the formation of ethoxyformic histidine. The reversal of inactivation by hydroxylamine and a pKa value of 7.0 obtained from the pH-rate profile for inactivation again confirmed the specificity of DEP for histidine. Stoichiometric results showed that two moles of histidine residue per mole of enzyme are essential for the activity of FAS.
Mukherjee
S
S
Department of Chemistry, Indian Institute of Technology, Kanpur, India.
Katiyar
S S
SS
D000818:Animals; D001665:Binding Sites; D010856:Columbidae / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004789:Enzyme Activation / Q000187:drug effects; D064429:Fatty Acid Synthases / Q000187:drug effects / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013997:Time Factors
D006898:Hydroxylamines; D019811:Hydroxylamine; D006639:Histidine; D009249:NADP; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D064429:Fatty Acid Synthases; D004047:Diethyl Pyrocarbonate