Use of Deuterium Kinetic Isotope Effects To Probe the Cryptoregiochemistry of Δ9 Desaturation

Abstract

The biological syn-dehydrogenation (desaturation) of fatty acids 1 as exemplified by the ∆9 desaturase-mediated transformation of stearoyl CoA (1) to give oleyl CoA (2) represents one of the more virtuosic displays of enzymatic selectivity. Two classes of desaturases catalyze this intriguing transformation: soluble plant enzymes containing a carboxylate-bridged, nonheme diiron center 2 and less well-characterized, nonheme iron, membrane-bound catalysts as represented by the ∆9 desaturase found in Saccharomyces cereVisiae 3 and rat liver. 4 In light of the mounting evidence 5 that desaturases and hydroxylases are structurally related at the protein level, we have adopted the view that desaturations are initiated by a hydrogen abstraction step similar to that proposed for biohydroxylation. Some of the possible subsequent steps to the olefin are outlined in Scheme 1. 6 We have focussed our attention on the ∆9 desaturase of S. cereVisiae and have tentatively placed the putative iron-oxo oxidizing species near C-9 of the substrate since this enzyme system consistently oxygenates 9-thia fatty acid analogues more efficiently than the corresponding 10-thia analogues. 12 In this communication, we report the results of a study in which we further investigate the cryptoregiochemistry of yeast ∆9 desaturation by measuring the deuterium isotope effect for each individual C-H bond cleavage. 13 In order to expedite our isotope effect study, we decided to run direct competition experiments involving methyl 7-thiastearate-9,9-d 2 (3-9,9-d 2 ) vs methyl 7-thiastearate (3) and methyl 7-thiastearate-10,10-d 2 (3-10,10-d 2 ) vs methyl 7-thiastearate (3). Use of methyl stearate-d 2 /d 0 mixtures would have complicated the analysis of the methyl oleate-d 1 /d 0 product due to mass spectral interference by endogenous (d 0 ) oleate. The sulfur atom was placed at position 7 in order to facilitate the synthesis of the deuterated substrates. We have shown previously that methyl 7-thiastearate (3) is converted to the corresponding thiaoleate product (4). 14 3-9,9-d 2 and 3-10,10-d 2 were synthesized in 10% and 8% overall yield, respectively, using well-known procedures as shown in Scheme 2. 15 The two deuterated substrates consisted entirely of dideuterated species (within experimental error) as determined by MS; the 1 H and 13 C NMR spectra were consistent with the location of deuterium label. 3 was available from our previous study. A ca. 1:1 mixture of each deuterated substrate and d 0 material (25 mg) was administered as 5% w/v ethanolic solutions to growing cultures (150 mL) of S. cereVisiae ATCC 12341 as previously described. 12 Each incubation was carried The putative radical intermediate 7 could follow at least three different pathways: one-electron oxidation/H + elimination (pathway a), 8a,b disproportionation (pathway b), 9 or a hydroxyl rebound 10a (SH2) 10b /fast Fe 3+promoted dehydration sequence (pathway c). In addition, the possibility that organoiron intermediates 11 (not shown) are involved in these reactions cannot be...

Desaturation and Hydroxylation

“…The intrinsic hydroxylation activity of desaturases with different regiospecificities makes these enzymes possible targets for future directed evolution experiments. Furthermore, the observation of low level hydroxylation activity at C-12 for the ⌬ 12 desaturase from A. thaliana, C-9 for the ⌬ 9 desaturase of S. cerevisiae, C-5 for the ⌬ 5 desaturase of B. subtilis, and C-15 for the -3 linoleate desaturase from flax suggests that these positions are the sites of initial oxidation of the desaturation reactions, corroborating the kinetic isotope effect studies of Buist and coworkers (19,(32)(33)(34).…”

ω‐Fluoro Thiafatty Acids: New Mechanistic Probes of Desaturase‐Mediated Reactions

“…It is clear that the presence of a substrate w-F-tag did not significantly affect the pronounced preference of the desaturase system to oxygenate 9-thia substrate analogues as we have previously documented (see Introduction). The ratio of 9-to 10-sulfoxidized products (1.6 : 1) obtained in this work compares favorably with the values obtained from both direct competition (2.1 : 1 [6]) and noncompetitive incubation of thiastearates (1.7 [5]). Good correlation between sulfoxidation ratios derived from experiments with nonfluorinated and w-fluorinated thiastearates have also been obtained for a soluble, plant D9-desaturase system [9] [15].…”

“…However, some important mechanistic information has been obtained on the structurally related yeast enzyme [4], which can be conveniently studied in vivo by an appropriate experimental design. A KIE study showed that the H-atom at C(9) of substrate is removed first in an isotopically sensitive step, followed by rapid cleavage of the CÀH bond at C(10) [5]. This result correlates well with the observation that desaturase-mediated oxo transfer occurs most efficiently when a S-atom occupies the 9-position of a thiastearoyl substrate (Scheme 1, b) [6]; the enantioselectivity of sulfoxidation matches the stereochemistry (syn, pro-R) of the corresponding parent reaction [7] [8].…”

Natural Deuterium Distribution in Fatty Acids Isolated from Peanut Seed Oil: A Site-Specific Study by Quantitative 2H NMR Spectroscopy

“…[30] The current data indicate that this overall isotope effect contains a significant secondary KIE component. A strong KIE has also been reported at C12 during D 12 desaturation.…”