Ph. Buist et B. Behrouzian, DECIPHERING THE CRYPTOREGIOCHEMISTRY OF OLEATE DELTA(12) DESATURASE -A KINETIC ISOTOPE EFFECT STUDY, Journal of the American Chemical Society, 120(5), 1998, pp. 871-876
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.