Δ6-desaturase: improved methodology and analysis of the kinetics in a multi-enzyme system

• Published in: Biochimica et biophysica acta, Protein structure and molecular enzymology Protein structure and molecular enzymology, 1996, Vol.1292 (1), p.120-132
• Main Authors: Ivanetich, Kathryn M., Bradshaw, Jean J., Ziman, Melanie R.
• Format: Article
• Language: English
• Subjects: Acyl-CoA synthetase; Computer modeling; Enzyme kinetics; Fatty acid desaturation; Linoleic acid; Lysophospholipid acyltransferase; Δ6-Desaturase
• ISSN: 0167-4838; 1879-2588
• DOI: 10.1016/0167-4838(95)00174-3

A new method of assay for the A6-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%) [l], and avoided the step of methylation of the saponified fatty acid substrate and product. Using this new method of assay, the kinetics of the Δ6-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 ± l.0 AM (0.5 mg microsomal protein/ml), which was comparable to added substrate concentrations (l.8 to 7.9 μM). The kinetics of the Δ6-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 l.5 μM and 0.063 nmol/min were calculated for the Δ6-desaturase, compared to K m and V max of 10.7 μM 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 Δ6-desaturase. These results have implications for kinetic analyses of all desaturases in microsomal systems.