Use of deuterium kinetic isotope effects to probe the cryptoregiochemistry of Δ9 desaturation

Full text of DOI:10.1021/ja953262n

J. Am. Chem. Soc. 1996, 118, 6295-6296
6295
Communications to the Editor
Scheme 1
Use of Deuterium Kinetic Isotope Effects To Probe
the Cryptoregiochemistry of ∆9 Desaturation
Peter H. Buist* and Behnaz Behrouzian
Ottawa-Carleton Chemistry Institute
Department of Chemistry, Carleton UniVersity
1125 Colonel By DriVe, Ottawa
Ontario, Canada, K1S 5B6
ReceiVed September 25, 1995
The biological syn-dehydrogenation (desaturation) of fatty
acids1 as exemplified by the ∆9 desaturase-mediated transfor-
mation 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, non-
heme diiron center2 and less well-characterized, nonheme iron,
membrane-bound catalysts as represented by the ∆9 desaturase
found in Saccharomyces cereVisiae3 and rat liver.4 In light of
the mounting evidence5 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-thia-
stearate-9,9-d2 (3-9,9-d2) vs methyl 7-thiastearate (3) and methyl
7-thiastearate-10,10-d2 (3-10,10-d2) vs methyl 7-thiastearate (3).
Use of methyl stearate-d2/d0 mixtures would have complicated
the analysis of the methyl oleate-d1/d0 product due to mass
spectral interference by endogenous (d0) 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
(1) Cook, H. In Biochemistry of Lipids and Membranes Vance, D. E.,
Vance, J. E., Eds.; The Benjamin Cummings Publishing Co. Ltd.: Menlo
Park, CA, 1985; pp 191-203.
(2) Fox, B. G.; Shanklin, J.; Somerville, C.; Munck, E. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 2486.
3-9,9-d2 and 3-10,10-d2 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 1H and 13C 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 d0 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
(3) Stukey, J. E.; McDonough, V. M.; Martin, C. E. J. Biol. Chem. 1990,
265, 20144.
(4) Enoch, H. G.; Catala, A.; Strittmatter, P. J. Biol. Chem. 1976, 251,
5095.
(5) (a) Fox, B. G.; Shanklin, J.; Ai, J.; Loehr, T. M.; Sanders-Loehr, J.
Biochemistry 1994, 33, 12776. (b) Shanklin, J.; Whittle, E.; Fox, B. G
Biochemistry 1994, 33, 12787. (c) Van de Loo, F. J.; Broun, P.; Turner, S.;
Somerville, C. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6743.
(6) The putative radical intermediate7 could follow at least three different
pathways: one-electron oxidation/H+ elimination (pathway a),8a,b dispro-
portionation (pathway b),9 or a hydroxyl rebound10a (SH2)10b/fast Fe3+
-
promoted dehydration sequence (pathway c). In addition, the possibility
that organoiron intermediates11 (not shown) are involved in these reactions
cannot be excluded at the present time.
(13) An overall, intermolecular deuterium isotope effect of 6.0 was
obtained using the closely related rat liver ∆9 desaturase and noncompetitive
comparison of oxidation rates for stearoyl CoA-9,9,10,10-d4 vs stearoyl
CoA .4
(14) Buist, P. H.; Dallmann, H. G.; Rymerson, R. R.; Seigel, P. M.; Skala,
P. Tetrahedron Lett. 1988, 29, 435. Our whole cell system allows us to use
thiastearate methyl esters which are presumably converted intracellularly
to the corresponding CoA thioesters prior to desaturation. The thiaoleate
products can then be isolated by a cell hydrolysis/methylation sequence.
The use of S. cereVisiae under conditions of adequate glucose supply
prevents extensive metabolism of the thiastearate substrates and/or the
thiaoleate products which in turn might lead to nondesaturase-related isotopic
fractionation.
(7) Very recent results from the Newcomb laboratory have suggested
that this species is not a true intermediate but, at least in the case of certain
cyclopropylcarbinyl radicals, behaves as “a component of a reacting
ensemble” with a very short lifetime of ca. 70 fs: Newcomb, M.;
Letadicbiadetti, F. H.; Chestney, D. L.; Roberts, E. S.; Hollenberg, P. F. J.
Am. Chem. Soc. 1995, 117, 12085.
(8) (a) Ortiz de Montellano, P. R. Trends. Pharmacol. Sci. 1989, 10,
354. (b) Collins, J. R.; Camper, D. L.; Loew, G. H. J. Am. Chem. Soc.
1991, 113, 2736.
(9) Akhtar, M.; Wright, J. N. Nat. Prod. Rep. 1991, 8, 527.
(10) (a) Groves, J. T.; McClusky, G. A.; White, R. E.; Coon, M. J.
Biochem. Biophys. Res. Commun. 1978, 81, 154. (b) Bowry, V. W.; Lusztyk,
J.; Ingold, K. U. J. Am. Chem. Soc. 1991, 113, 5699.
(15) The tosylate of decan-1-ol-1,1-d2 was prepared by reduction of
decanoic acid using LiAlD4, followed by treatment with TsCl. 1-bro-
mononane-1,1,-d2 was similarly available by LiAlD4 reduction of nonanoic
acid followed by bromination of the resultant alcohol using PBr3.
(11) Blackburn, J. M.; Sutherland, J. D.; Baldwin, J. E. Biochemistry
1995, 34, 7548.
(12) Buist, P. H.; Marecak, D. M. J. Am. Chem. Soc. 1992, 114, 5073.
S0002-7863(95)03262-8 CCC: $12.00 © 1996 American Chemical Society

6296 J. Am. Chem. Soc., Vol. 118, No. 26, 1996
Communications to the Editor
Table 1. Intermolecular Isotopic Discrimination in ∆9 Desaturation of Methyl 7-Thiastearate-9,9-d2 and Methyl 7-Thiastearate-10,10-d2
isotopic composition (mol %)a
substratesb
products
expt
d0
9-d2
10-d2
d0
9-d1
10-d1
KIE
1
2
3
47.2 ( 0.7
47.0 ( 0.7
46.8 ( 0.3
52.8 ( 0.7
53.0 ( 0.8
53.2 ( 0.3
86.8 ( 0. 4
86.1 ( 0. 8
86.0 ( 0.4
13.2 ( 0.4
13.9 ( 0.8
14.0 ( 0.4
7.4 ( 0.3
7.0 ( 0.4
7.0 ( 0.2
(7.1 ( 0.2)c
1.13 ( 0.03
0.98 ( 0.01
0.98 ( 0.02
(1.03 ( 0.09)d
4
5
6
49.2 ( 0.4
50.9 ( 0.3
49.7 ( 0.4
50.8 ( 0.4
49.1 ( 0.3
50.3 ( 0.4
52.3 ( 0.8
50.3 ( 0.1
49.2 ( 0.6
47.7 ( 0.8
49.7 ( 0.1
50.8 ( 0.6
a The mol % of each isotopic species is given as an average value based on two to three GC/MS runs. b The six starting mixtures were individually
prepared. c The average KIE at C-9 ( standard deviation. d The average KIE at C-10 ( standard deviation.
Scheme 2
out three times. After 24 h of growth, the cellular fatty acids
(ca. 30 mg) were isolated from the centrifuged cells by a
standard hydrolysis (1 M 50% ethanolic KOH)/methylation
(CH2N2) sequence, and the deuterium content of thiaoleate
products (<10% of the total fatty acid fraction) was assessed
by GC/MS (30 m DB-5 capillary column, temperature pro-
grammed from 120 °C, (held, 2 min) to 320 °C at 10 °C/min;
MS scan rate: 0.5 s/decade; 5-8 scans per GC peak; the
integrated intensities of the individual ions in the molecular ion
cluster were recorded using a SUN SPARC I workstation
equipped with Kratos Mach 3 software and have been corrected
for natural isotopic abundances.) Product kinetic isotope effects
(KIEs) (kH/kD) were calculated using the ratio [% d0 (product)/
% d1 (product)] / [% d0 (substrate) / % d2 (substrate)]. In this
manner, a large primary deuterium isotope (7.1 ( 0.2, average
of 3 experiments)16 was determined for the carbon-hydrogen
thought to be initiated by hydrogen abstraction at C-4.18 It is
also interesting to note that in very early experiments involving
9- and 10-monotritiated stearates and a bacterial ∆9 desaturase,
a kinetic isotope effect at C-9 but not at C-10 was observed.19
Our results are not consistent with a desaturase mechanism
involving a synchronous removal of the 9,10-hydrogenssa
possibility which has been raised previously.20,21
In conclusion, we believe that the methodology described in
this paper will prove very useful in the study of other desaturases
such as the recently discovered soluble, diiron plant ∆9
desaturases for which no mechanistic information is currently
available.22
bond cleavage at C-9, while the C-10- H bond-breaking step
was shown to be essentially insensitive to deuterium substitution
(KIE ) 1.03 ( 0.09, average of 3 experiments). The analytical
data is displayed in Table 1.
These results represent the first direct determination of the
KIE17 on each of the two C-H bond cleavage steps involved
in fatty acid desaturation and strongly suggest that the yeast
∆9 desaturase initiates oxidation at carbon 9 in a relatively slow,
isotopically sensitive step in accordance with our mechanistic
model. The second C-H cleavage appears to be fast as might
be expected for any of the three pathways shown in Scheme 1.
A similar pattern of isotope effects (one large (5.58, C-4) and
Acknowledgment. We thank NSERC for financial support of this
work. The technical assistance of Dr. Clem Kazakoff (University of
Ottawa) is greatly appreciated.
one small (1.62, C-5)) was observed for the cytochrome P450
-
mediated 4,5-dehydrogenation of valproic acid (5)sa reaction
(16) A small proportion (<10%) of the observed isotope effect may be
due to an R-secondary isotope effect. Cf.: Jones, J. P.; Trager, W. F. J.
Am. Chem. Soc. 1987, 109, 2171.
(17) The magnitude of the primary deuterium isotope effect at C-9 must
be regarded as a lower limit for the “intrinsic” kinetic isotope effect since
our value is derived via an intermolecular competition experiment and is
therefore subject to partial masking by other enzymic steps. Large “intrinsic”
primary deuterium isotope effects are not uncommon for biological
hydrocarbon activations thought to involve iron-oxo species. Cf. ref 16.
JA953262N
(18) Rettie, A. E.; Boberg, M.; Rettenmeier, A. W.; Baillie, T. A. J.
Biol. Chem. 1988, 263, 13733.
(19) Schroepfer, G. J.; Bloch, K. J. Biol. Chem. 1965, 240, 54.
(20) Morris, L. J.; Harris, R. V.; Kelly, W.; James, A. T. Biochem. J.
1968, 109, 673.
(21) Johnson, A. R.; Gurr, M. I. Lipids 1971, 6, 78.
(22) Feig, A. L.; Lippard, S. J. Chem. ReV. 1994, 94, 759.