Acyl-CoA synthetase activity of rat heart mitochondria. Substrate specificity with special reference to very-long-chain and isomeric fatty acids.

The acyl-CoA synthetase (acid: CoA ligase (AMP-forming), EC 6.2.1.3) activity of rat heart has been measured in fatty acid-depleted fractions of mitochondria, microperoxisomes and microsomes. The assay was based on (i) the measurement of the reaction product AMP by high-performance liquid chromatography or (ii) a coupled reaction in which the intramitochondrial (matrix) CoASH is the final acyl acceptor and the redox state of the flavoproteins in the acyl-CoA dehydrogenase pathway is used to determine the intramitochondrial level of acyl-CoA. This spectrophotometric method was also used to estimate the 'outer' carnitine long-chain acyltransferase (palmitoyl-CoA:L-carnitine O-palmitoyltransferase, EC 2.3.1.21) activity. Comparison of the distribution of long-chain acyl-CoA synthetase activity and marker enzymes in the various subcellular fractions revealed that the synthetase activity is exclusively localized in the mitochondrial fraction. Experimental evidence is presented in support of the conclusion that the chain-length specificity of saturated and monounsaturated fatty acids (16:1-22:1) for the acyl-CoA synthetase is mainly determined by the availability of the fatty acid at the active site, which is largely determined by the affinity of binding of fatty acids to the bulk phase of the mitochondrial phospholipids. Among the 22:1 isomers, 22:1(11) (cis) (cetoleic acid) revealed a slightly higher activity (1.4-fold) than 22:1(13) (cis) (erucic acid). The polyunsaturated fatty acids tested were rather poor substrates. Using isolated intact mitochondria and 16:0 or 22:1(13) (cis) as the substrates, it was found that the initial rate of the 'outer' long-chain acyltransferase activity was approximately four times higher than that of the long-chain acyl-CoA synthetase. The data support the hypothesis that the long-chain acyl-CoA synthetase reaction is rate-limiting in the sequence of coupled reactions leading to beta-oxidation in the mitochondrial matrix.