The reaction of fatty acyl thiolesters with proteins.

Abstract

The enzyme fraction precipitated from rat liver cell sap at pH 5 catalyzes the formation of fatty acyl coenzyme A thiolesters. During the incubation, about 3% of the labeled fatty acid appears covalently bound to protein. The fatty acyl CoA esters were the intermediates in the reaction. The covalent linkage of the fatty acid to the protein was not enzymatic, since labeled palmityl CoA reacted directly with protein. Proof for the covalent nature of the binding was obtained in three ways: (a) the label was not removed by a series of solvent extractions which removed free fatty acid or fatty acyl CoA, (b) labeled palmityl peptides were isolated from peptic digests of labeled protein, (c) peptides isolated from protein labeled with [1‐14C]palmitate were not decarboxylated by azide in the Schmidt reaction.Covalent labeling of the pH 5 enzyme protein was extensive only with fatty acids above 10 carbon atoms. Acetyl CoA gave less than 10% of the reaction given by palmityl CoA. These results suggested a preliminary binding of the hydrocarbon tail of the CoA esters with hydrophobic regions of the protein.Evidence for the involvement of protein hydroxyl and amino groups in the reaction was obtained. Reduction of pronase peptides of protein labeled with palmitic acid with LiAlH4 gave rise to labeled cetyl alcohol. SH Groups were not involved as judged by the failure of neutral hydroxylamine to remove the label and by the fact that gamma globulin, which has no free SH groups, was capable of reacting. Acid or alkaline hydrolysis at 100° removed less than half of the label, suggesting the presence of amide bonds. Blockage of amino groups by forming the picryl derivative of the protein greatly reduced the extent of the reaction.