DECIPHERING THE CRYPTOREGIOCHEMISTRY OF OLEATE DELTA(12) DESATURASE -A KINETIC ISOTOPE EFFECT STUDY

The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in linoleic acid biosynthesis were d etermined using a suitably transformed strain of Saccharomyces cerevis iae containing a functional oleate Delta(12) desaturase from Arabidops is thaliana. Mass spectral analysis of the methyl 7-thialinoleate frac tion obtained from competition experiments involving methyl 7-thiastea rate, methyl [12,12-H-2(2)]-7-thiastearate and methyl [13,13-H-2(2)]-7 -thiastearate showed that cleavage of the C-12-H bond is very sensitiv e to isotopic substitution (k(H)/k(D) = 7.3 +/- 0.4) while a negligibl e isotope effect (k(H)/k(D) = 1.05 +/- 0.04) was observed for the C-13 -H bond breaking step. This result strongly suggests that the site of initial oxidation for Delta(12) desaturation is at C-12. The possible relationship between castor oleate 12-hydroxylase and microsomal Delta (12) oleate desaturases is discussed in the context of a common mechan istic paradigm. Our methodology may be also be useful in deciphering t he cryptoregiochemistry of other desaturase systems.