The presence of essential arginine residues at the NADPH-binding sites of beta-ketoacyl reductase and enoyl reductase domains of the multifunctional fatty acid synthetase of chicken liver.
Treatment of chicken liver fatty acid synthetase with the arginine-specific reagent phenylglyoxal resulted in the pseudo-first-order loss of synthetase, beta-ketoacyl reductase and enoyl reductase activities. The sum of the second-order rate constants for the two reductase reactions equalled that for the synthetase reaction, suggesting that inactivation of either reductase was responsible for the loss of fatty acid synthetase activity. Double-log plots of pseudo-first-order rate constant versus reagent concentration yielded straight lines with slopes of unity for all three activities tested, suggesting the reaction of one reagent molecule in the inactivation process. In parallel experiments, complete inactivation of synthetase activity was accompanied by the incorporation of 4.5 [14C]phenylglyoxal, and the loss of 2.3 arginine residues per subunit. Reaction of essential sulfhydryl groups was not involved, since inactivation by phenylglyoxal was unaffected by reversible protection of these groups with 5,5'-dithiobis(2-nitrobenzoic acid). Inactivation of all three activities by phenylglyoxal was prevented by saturating amounts of the coenzyme NADPH, or its analogs 2',5'-ADP and 2'-AMP, but not by the corresponding nucleotides containing only the 5'-phosphate. Conversely, the ability of this enzyme to bind NADPH was abolished upon inactivation. These results are consistent with the presence of an essential arginine residue at the binding site for the 2'-phosphate group of NADPH at each of the two reductase domains of the multifunctional fatty acid synthetase subunit.
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001120:Arginine; D001665:Binding Sites; D002645:Chickens; D044943:Fatty Acid Desaturases / Q000378:metabolism*; D064429:Fatty Acid Synthases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D010658:Phenylglyoxal / Q000494:pharmacology; D013329:Structure-Activity Relationship
D009249:NADP; D001120:Arginine; D000429:Alcohol Oxidoreductases; C021461:beta-ketoacyl thioester reductase; D044943:Fatty Acid Desaturases; D064429:Fatty Acid Synthases; D010658:Phenylglyoxal