Mechanism of fatty acid desaturation in the green alga Chlorella vulgaris

The hypothesis that the Delta (9) desaturase of Chlorella vulgaris might op erate by a synchronous mechanism has been tested using a kinetic isotope ef fect (KIE) approach. Thus the intermolecular primary deuterium KIE on the i ndividual C-H bond cleavage steps involved in Delta (9) desaturation have b een determined by incubating growing cultures of C. vulgaris (strain 211/8K ) with mixtures of the appropriate regiospecifically deuterated fatty acid analogues. Our analysis shows that the introduction of a double bond betwee n C-9 and C-10 occurs in two discrete steps as the cleavage of the C9-H bon d is very sensitive to isotopic substitution (k(H)/k(d) = 6.6 +/- 0.3) wher eas a negligible isotope effect (k(H)/k(d) = 1.05 +/- 0.05) was observed fo r the C10-H bond-breaking step. Similar results were obtained for linoleic acid biosynthesis (Delta (12) desaturation). These data clearly rule out a synchronous mechanism for these reactions.