Chemical modification of an essential lysine at the active site of enoyl-CoA reductase in fatty acid synthetase

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Fatty acid synthetase from goose uropygial gland was inactivated by treatment with pyridoxal 5′-phosphate. Malonyl-CoA and acetyl-CoA did not protect the enzyme whereas NADPH provided about 70% protection against this inactivation. 2′-Monophospho-ADP-ribose was nearly as effective as NADPH while 2′-AMP, 5′-AMP, ADP-ribose, and NADH were ineffective suggesting that pyridoxal 5′-phosphate modified a group that interacts with the 5′-pyrophosphoryl group of NADPH and that the 2′-phosphate is necessary for the binding of the coenzyme to the enzyme. Of the seven component activities catalyzed by fatty acid synthetase only the enoyl-CoA reductase activity was inhibited. Inactivation of both the overall activity and enoyl-CoA reductase of fatty acid synthetase by this compound was reversed by dialysis or dilution but not after reduction with NaBH 4. The modified protein showed a characteristic Schiff base absorption (maximum at 425 nm) that disappeared on reduction with NaBH 4 resulting in a new absorption spectrum with a maximum at 325 nm. After reduction the protein showed a fluorescence spectrum with a maximum at 394 nm. Reduction of pyridoxal phosphate-treated protein with NaB 3H 4 resulted in incorporation of 3H into the protein and paper chromatography of the acid hydrolysate of the modified protein showed only one fluorescent spot which was labeled and ninhydrin positive and had an R f identical to that of authentic N 6-pyridoxyllysine. When [4- 3H]pyridoxal phosphate was used all of the 3H, incorporated into the protein, was found in pyridoxyllysine. All of these results strongly suggest that pyridoxal phosphate inhibited fatty acid synthetase by forming a Schiff base with the ϵ-amino group of lysine in the enoyl-CoA reductase domain of the enzyme. The number of lysine residues modified was estimated with [4- 3H]pyridoxal-5′-phosphate/NaBH 4 and by pyridoxal-5′-phosphate/NaB 3H 4. Scatchard analysis showed that modification of two lysine residues per subunit resulted in complete inactivation of the overall activity and enoyl-CoA reductase of fatty acid synthetase. NADPH prevented the inactivation of the enzyme by protecting one of these two lysine residues from modification. The present 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.