Dihydroceramide Delta(4) desaturase initiates substrate oxidation at C-4

The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in dihydroceramide Delta (4) desaturation have been determined for the first time by incubating rat liver microsomes with 1:1 mixtures of nonlabeled substrate and the appropriate regiospecifically dideutelatred analogue. Analysis of the enzymatic products via gas chromat ography coupled to mass spectrometry showed that the introduction of the (E ) double bond between C-4 and C-5 occurs in two discrete steps: cleavage of the C-4-H bond was found to be very sensitive to isotopic substitution (k( H)/k(D) = 8.0 +/- 0 8). while a negligible isotope effect (k(H)/k(D) = 1.02 +/- 0.07) was observed for the C-5-H bond-breaking step. According to a me chanistic model that we have previously proposed, these results suggest tha t initial oxidation for this desaturation reaction occurs at C-4. This find ing correlates nicely with the observation that 4-hydroxylated products are produced from a similar substrate by a closely related oxidative enzyme in yeast.