GROVE*, T J; SIDELL, B D: Examining Substrate Specificity of Fatty Acyl CoA Synthetase in Notothenioid Fishes

Oxidative muscle tissues of Antarctic notothenioid fishes are more dependent upon lipids than carbohydrates as their main fuel for aerobic energy metabolism. These tissues further oxidize mono-unsaturated fatty acids more readily than saturated fatty acids. We hypothesize that fatty acyl CoA synthetase (FACS; EC 6.2.1.3) of Antarctic notothenioid fishes exhibits specificity for activating long-chain unsaturated fatty acids over saturated fatty acids, thus targeting monoenes for preferential oxidation in the mitochondria. Full-length FACS cDNAs from oxidative skeletal muscles of Chaenocephalus aceratus, Gobionotothen gibberifrons, and Notothenia coriiceps were previously identified by RACE PCR and sequenced. We have extended our study to include two sub-Antarctic notothenioids, Eliginops maclovinus and Notothenia angustata. Preliminary sequence analysis between Antarctic notothenioids and E. maclovinus shows 94-95% identity. We have also adapted an enzyme-linked continuous spectrophotometric enzyme assay to examine the functionality and substrate specificity of FACS at the physiological temperatures of these fishes. Using this assay, we are examining substrate specificity of FACS by measuring the activity of FACS in the presence of fatty acids with different chain lengths and degrees of saturation. If, as we hypothesize, FACS exhibits specificity for long-chain mono-unsaturated fatty acids, then further studies using site-directed mutagenesis will be conducted to identify whether amino acid substitutions within the fatty acid-binding pocket play important roles in substrate specificity of the enzyme. Supported by NSF grants OPP 94-21657 and OPP 99-09055 to BDS.