Delta 6-desaturase: Improved methodology and analysis of the kinetics in a multi-enzyme system

A new method of assay for the Delta 6-desaturation of linoleic acid was developed. This method, which uses HPLC for separation of the fatty acid substrate and product, exhibited a lower coefficient of variation (0.3%) than the reported TLC method (3.5%) [1], and avoided the step of methylation of the saponified fatty acid substrate and product. Using this new method of assay, the kinetics of the Delta 6-desaturase in a multi-enzyme system were analysed. A number of factors that could have striking effects on desaturase kinetics were investigated, including the effect of (i) endogenous microsomal linoleic acid on total substrate concentration, and (ii) the pre-reaction catalysed by acyl-CoA synthetase and competing reactions catalysed by lysophospholipid acyltransferase and acyl-CoA hydrolase, Endogenous free linoleate in the hepatic microsomes was found to be 2.9 +/- 1.0 M (0.5 mg microsomal protein/ml), which was comparable to added substrate concentrations (1.8 to 7.9 mu M). The kinetics of the Delta 6-desaturase were dissected from the kinetics of the above mentioned pre-reaction and competing reactions through a combination of experimental approaches and computer modeling. From computer modeling, a K-m and V-max of 1.5 mu M and 0.063 nmol/min were calculated for the Delta 6-desaturase, compared to K-m and V-max of 10.7 mu M and 0.08 nmol/min calculated directly from data uncorrected for endogenous substrate. It was concluded that lysophospholipid acyltransferase, acyl-CoA synthetase and endogenous linoleic acid significantly affect the kinetic measurements of hepatic microsomal Delta 6-desaturase. These results have implications for kinetic analyses of all desaturases in microsomal systems.