Kinetic Studies on the Specificity of Long Chain Acyl Coenzyme A Synthetase from Rat Liver Microsomes

The kinetics of activation of unsaturated and saturated fatty acids by long chain acyl-CoA synthetase from rat liver microsomes has been studied with a method of selective extraction of free fatty acids based on the insolubility of acyl-CoA in diethyl ether. All unsaturated fatty acids with a double bond in Δ9 position (palmitoleate, oleate, linoleate, linolenate) have values of Km in the same range (1.39 to 2.22 µm) and values of Vmax which vary from 9.09 to 18.52 µmoles per hour per mg of protein. These fatty acids are competitive inhibitors of the activation of each other. Each of these fatty acids has the same Ki (1.23 µm) for the inhibition of linolenic acid activation. Some of the metabolites of linoleic acid, eicosa-11,14-dienoic, eicosa-8,11,14-trienoic, and eicosa-5,8,11,14-tetraenoic acids, and of linolenic acid, eicosa-11,14,17-trienoic acid, are also competitive inhibitors of the activation of palmitoleic acid: the two eicosatrienoic acids are the strongest inhibitors (Ki = 4.2 µm), followed by the eicosatetraenoic acid (Ki = 5.0 µm), and by the eicosadienoic acid (Ki = 7.0 µm). The Km values for CoA in the activation of palmitic and oleic acids are, respectively, 7.2 and 9.4 µm. The Km for activation of palmitic acid is 2.78 µm with a Vmax = 14.29 µmoles per hour per mg of protein. Palmitic acid is also a competitive inhibitor of linolenic acid activation with the same Ki (1.23 µm) as that of unsaturated fatty acids with a Δ9 double bond. Similarly palmitic and stearic acids are competitive inhibitors of palmitoleic acid activation (Ki = 2.4 and 17.3 µm, respectively). These results lead to the theoretical conclusion that there is probably only one long chain acyl-CoA synthetase which contains several active sites: a main site which may bind the carboxyl group, and therefore would be the same for all substrates, and secondary sites (or spatial configuration) to accommodate the different types of fatty acid molecules.