SYNTHESIS OF AN UNSATURATED FATTY-ACID ANALOG (18-(4'-AZIDO-2'-HYDROXYBENZOYLAMINO)-OLEIC ACID) AND ITS INTERACTION WITH LYSOPHOSPHATIDYLCHOLINE - ACYL-COA-O-ACYLTRANSFERASE

Acylation/deacylation reactions represent a basic requirement of trigl yceride as well as phospholipid metabolism, and maintenance of membran e lipid composition, In order to examine enzymes participating in thes e pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acid s or acyl-CoAs. For the synthesis of omega-amino-oleic acid, coupling two bifunctional C-9-components was used, The described synthesis sche me is also suited for the specific generation of other fatty acid anal ogues with distinct positions of the double bond, The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [ EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsa turated fatty acyl-CoAs. It could be shown that the photolabel, esteri fied with coenzyme A, acts in the dark as a reversible inhibitor of th e enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis, Several pr oteins could be specifically visualized using the iodinated analogue.i r The data indicate that this new photoaffinity label may have applica tion to identify and characterize Lipid biosynthetic enzymes using uns aturated fatty acids as well as acyl-CoA binding proteins and the acti ve site of these proteins.