Dihydroceramide Δ4 desaturase initiates substrate oxidation at C-4

Article abstract of DOI:10.1021/ja010088w

The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps involved in dihydroceramide Δ⁴ desaturation have been determined for the first time by incubating rat liver microsomes with 1:1 mixtures of nonlabeled substrate and the appropriate regiospecifically dideuterated analogue. Analysis of the enzymatic products via gas chromatography 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 C₄-H bond was found to be very sensitive to isotopic substitution (k_H/k_D= 8.0 ± 8.0), while a negligible isotope effect (k_H/k_D= 1.02 ± 0.07) was observed for the C₅-H bond-breaking step. According to a mechanistic model that we have previously proposed, these results suggest that initial oxidation for this desaturation reaction occurs at C-4. This finding correlates nicely with the observation that 4-hydroxylated products are produced from a similar substrate by a closely related oxidative enzyme in yeast.

Full text of DOI:10.1021/ja010088w

4382
J. Am. Chem. Soc. 2001, 123, 4382-4385
Dihydroceramide ∆4 Desaturase Initiates Substrate Oxidation at C-4
Christopher K. Savile,‡ Gemma Fabria`s,*,§ and Peter H. Buist*,‡
Contribution from the Department of Chemistry, Ottawa-Carleton Chemistry Institute, Carleton UniVersity,
1125 Colonel By DriVe, Ottawa, Ontario, Canada K1S 5B6, and Department of Biological Organic
Chemistry, IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, Spain
ReceiVed January 9, 2001
Abstract: The intermolecular primary deuterium isotope effects on the individual C-H bond cleavage steps
involved in dihydroceramide ∆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 dideuterated analogue.
Analysis of the enzymatic products via gas chromatography 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 C4-H
bond was found to be very sensitive to isotopic substitution (kH/kD ) 8.0 ( 0.8), while a negligible isotope
effect (kH/kD ) 1.02 ( 0.07) was observed for the C5-H bond-breaking step. According to a mechanistic
model that we have previously proposed, these results suggest that initial oxidation for this desaturation reaction
occurs at C-4. This finding correlates nicely with the observation that 4-hydroxylated products are produced
from a similar substrate by a closely related oxidative enzyme in yeast.
Introduction
The essential role of sphingolipids in the cell biology of both
mammalian and yeast cells has been well documented.1 In
mammals, ceramide 1 (an important member of the sphingolipid
family) acts as a second messenger in mediating antimitogenic
effects, such as cell growth supression, cell cycle arrest, and
programmed cell death or apoptosis. Ceramide is also an
important regulator of cellular responses to stress. Sphingosine-
1-phosphate, a deacylated ceramide derivative, is a potent
mitogen that regulates mobilization of calcium, cell growth,
differentiation, survival, and motility. In Saccharomyces cer-
eVisiae, sphingolipids and their derivatives are known to
participate in such functions as signal transduction during
response to heat stress, regulation of calcium homeostasis,
calcium-mediated signaling, and regulation of the cell cycle.2
Interestingly, some activities of mammalian sphingolipids
depend critically3 on the presence of an (E)-4,5 double bond in
the long chain base structure of 1. This functional group is
formed by an unusual regio- and stereoselective dehydrogenation
process catalyzed by a membrane-bound enzyme known as
dihydroceramide desaturase.4-6 Biochemical studies suggest that
this enzyme is closely related to a 4-hydroxylase which is
responsible for the biosynthesis of phytoanalogues of ceramide
1 such as 4-hydroxysphinganine 2.7 Indeed, desaturation can
be regarded as a mechanistic variation of hydroxylation and a
generic scheme linking the two pathways has been proposed.8
This features a common, initial C-H activation step to form a
very short-lived radical intermediate or its organoiron equivalent
(not shown)9 which can collapse rapidly to give either alcohol
or olefin (Scheme 1). The relative spatial relationship of the
putative diiron oxidant10a to substrate is thought to be primarily
* Address correspondence to this author.
‡ Carleton University.
§ IIQAB-CSIC.
(1) Hannun, Y. A. In Sphingolipid mediated signal transduction; Hannun,
Y. A., Ed.; R. G. Landes Company: Austin, TX, 1997; pp 1-15.
(2) Dickson, R. C. Annu. ReV. Biochem. 1998, 67, 27-48.
(3) He, L.; Byun, H.; Smit, J.; Wilschut, J.; Bittman, R. J. Am. Chem.
Soc. 1999, 121, 3897-3903.
(8) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1998, 120, 871-
876.
(4) Michel, C.; van Echten-Deckert, G.; Rother, J.; Sandhoff, K.; Wang,
E.; Merrill, A. H., Jr. J. Biol. Chem. 1997, 272, 22432-22437.
(5) Geeraert, L.; Mannaerts, G. P.; Van Veldhoven, P. P. Biochem. J.
1997, 327, 125-132.
(9) The precise nature of the intermediates in C-H activation reactions
of this type remains obscure. Recently, Groves et al. have obtained some
evidence for discrete carbon-centered radical intermediates in C-H activa-
tion reactions catalyzed by this class of enzyme: Austin, R. N.; Chang, H.;
Zylstra, G.; Groves, J. T. J. Am. Chem. Soc. 2000, 122, 11747-11748.
(10) (a) Shanklin, J.; Achim, C.; Schmidt, H.; Fox, B. G.; Munck, E.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2981-2986. (b) Shanklin, J.; Cahoon,
E. B. Annu. ReV. Plant Physiol. Plant Mol. Biol. 1998, 49, 611-641.
(6) Causeret, C.; Geeraert, L.; VanderHoven, G. P.; Mannaerts, G.; Van
Veldhoven, P. P. Lipids 2000, 35, 1117-1125.
(7) Grilley, M. M.; Stock, S. D.; Dickson, R. C.; Lester, R. L.; Takemoto,
J. Y. J. Biol. Chem. 1998, 273, 11062-11068.
10.1021/ja010088w CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/24/2001

Dihydroceramide ∆4 Desaturase Initiates Substrate Oxidation
J. Am. Chem. Soc., Vol. 123, No. 19, 2001 4383
All reagents and starting materials were purchased from Sigma-
Aldrich Canada (Oakville, Canada) and used without purification.
Tetrahydrofuran (THF), toluene, and diethyl ether (Et2O) were freshly
distilled from Na-benzophenone ketyl. All air- and moisture-sensitive
reactions were performed under N2. Organic extracts were typically
shaken with saturated NaCl and dried over MgSO4, and solvents were
evaporated in vacuo on a Bu¨chi RE 111 Rotavapor. [1,1-2H2]-1-
Bromopentadecane was prepared by reduction14 of pentadecanoic acid
with LiAlD4 followed by treatment with HBr/H2SO4.15 [2,2-2H2]-1-
Bromopentadecane was prepared by R-exchange of methyl pentade-
canoate with Na in MeOD16 followed by reduction with LiAlH414 and
treatment with HBr/H2SO4.15
Scheme 1
All buffers and salts were purchased from Merck (Darmstadt,
Germany); NADH, BSA, and other biochemicals were purchased from
Sigma-Aldrich Qu´ımica (Madrid, Spain). Protein concentrations were
measured by the method of Bradford17 using bovine serum albumin as
standard protein. Male Sprague-Dawley rats were supplied by the
animal breeding facility of the Centro de Investigacio´n y Desarrollo
(Barcelona, Spain).
responsible for the control of the reaction outcome.10b,11 Minor
changes in substrate structure can also effect a switch in the
reaction pathway.12
Synthesis of Substrates. D-erythro-Sphinganine was prepared es-
sentially as previously described18,19 by Grignard addition20 of penta-
decylmagnesium bromide to the Garner aldehyde.21 The deuterated
analogues were synthesized in similar fashion by using the appropriately
deuterated Grignard reagent. After deketalization of the Grignard adduct
with Amberlyst 15 catalyst,19 the resultant erythro derivative was freed
of the unwanted threo diastereomer by flash silica gel chromatography,
using hexane/EtOAc (3:1 v/v) as eluent. Cleavage19 of the carbamate
moiety with 1 N HCl in dioxane afforded D-erythro-sphinganine. The
analytical data are given below:
Given the obvious importance of dihydroceramide ∆4 de-
saturase, the efforts devoted to its purification, and the ongoing
search for inhibitors of this enzyme,13 an examination of the
mechanism of ∆4 desaturation is a worthy research objective.
In particular, we were interested in determining which methylene
group is attacked first in this enzymatic reaction since this is a
key determinant for the regiochemical outcome. Here, we report
the results of the first detailed mechanistic study of dihydro-
ceramide ∆4 desaturase in which we show that the oxidation of
substrate is initiated at C-4.
1
D-erythro-Sphinganine: mp 92-94 °C (lit.22 mp 85-88 °C); H
NMR (N-acetyl derivative, 400 MHz) δ 0.89 (t, J ) 6.7, 3H,
-CH2CH3), 1.28 (br s, 26 H, -(CH2)13CH3), 1.37 (m, 1H, C(4)-H),
1.51 (m, 1H, C(4)-H), 1.97 (s, 3H, -C(O)CH3), 3.59 (ddd, J ) 6.6,
2.7, 8.6, 1H, C(3)-H), 3.66 (dd, J ) 6.3, 11.3, 1H, C(1)-H), 3.70 (dd,
J 4.2, 11.3, 1H, C(1)-H), 3.81 (ddd, J ) 4.2, 6.3, 6.4, 1H, C(2)-H);
13C NMR (N-acetyl derivative, 100 MHz) δ 173.37 (CdO), 72.40 (C-
3), 62.16 (C-1), 57.03 (C-2), 34.83 (C-4), 33.08 (C-16), 30.79, 30.76,
30.48 (C-6 to C-15), 26.85 (C-5), 23.74 (C-2′), 22.77 (C-17), 14.44
(C-18); MS (N-octanoyl derivative, EI, 70 eV) m/z 556 (M+ - CH3),
468 (M+ - (CH3)3Si-O-CH2), 313 [((CH3)3Si-O-CH(CH2)14CH3)+],
258 [((CH3)3Si-O-CH2CH-NH-C(O)(CH2)6CH3)+].
Materials and Methods
General Methods. 1H (400 MHz) and 13C NMR (100 MHz) spectra
were obtained on a Bru¨ker AMX 400 spectrometer with the use of
dilute CDCl3 or CD3OD solutions. Chemical shifts are expressed in
ppm (δ) and are referenced to tetramethylsilane. J-values are reported
in hertz (Hz). Deuterium isotope effects on 13C NMR shifts were
estimated by running spectra of mixtures (1:2) of labeled and unlabeled
material. Mass spectra were obtained by GC/MS by using a Fisons
MD-800 mass spectrometer coupled to a Fisons 8000 series GC
equipped with a nonpolar Hewlett-Packard HP-1 capillary column (30
m × 0.20 mm), temperature programmed from 150 to 320 °C at 6
°C/min. Analyses were carried out under selected ion monitoring (SIM)
mode. The deuterium content was estimated by using a dwell time of
0.02 s which resulted in 5-8 scans per GC peak; the integrated
intensities of the individual ions in the pertinent ion cluster were
recorded by using Lab Base software and have been corrected for
natural isotopic abundances. Care was taken to include the entire GC
peak in the integration procedure to prevent errors due to fractionation
of isotopic species during chromatography. Isotopic ratios were
determined by using the following ions: m/z 313, ((CH3)3Si-O-CH-
(CH2)14CH3)+ (N-octanoyl-D-erythro-sphinganine 3), and m/z 311,
((CH3)3Si-O-CHCHdCH-(CH2)12CH3)+ (N-octanoyl-D-erythro-
sphingosine 4). Each mass spectral measurement was repeated a
minimum of three times to obtain an average value.
D-erythro-[4,4-2H2]Sphinganine was synthesized from [1,1-2H2]-1-
bromopentadecane. The spectral data of the title compound (N-acetyl
derivative) were similar to those of the unlabeled parent except for 1H
NMR (400 MHz) 1.37, 1.51 (m, 2H, C(4)-H2, absent), 3.58 (d, J )
6.7, 1H, C(3)-H); 13C NMR (100 MHz) 72.30 (C-3, upfield â-isotope
shift (0.11 ppm)), 57.01 (C-2, upfield γ-isotope shift (0.02 ppm)), 34.83
(C-4, absent), 26.65 (C-5, upfield â-isotope shift (0.19 ppm)); MS (N-
octanoyl derivative, EI, 70 eV) m/z 558 (M+ - CH3), 470 (M+
-
(CH3)3Si-O-CH2), 315 [((CH3)3Si-O-CHCD2(CH2)13CH3)+], 258
[((CH3)3Si-O-CH2CH-NH-C(O)(CH2)6CH3)+].
D-erythro-[5,5-2H2]Sphinganine was synthesized from [2,2-2H2]-1-
bromopentadecane. The spectral data of the title compound (N-acetyl
derivative) were similar to those of the unlabeled parent except for 1H
NMR (400 MHz) 1.37 (dd, J ) 13.8, 9.2, 1H, C(4)-H), 1.51 (dd, J )
3.3, 13.8, 1H, C(4)-H); 13C NMR (100 MHz) δ 72.38 (C-3, upfield
γ-isotope shift, (0.02 ppm)), 34.64 (C-4, upfield â-isotope shift (0.19
ppm)), 26.85 (C-5, absent); MS (N-octanoyl derivative, EI, 70 eV) m/z
Flash chromatography with silica gel (230-400 mesh) was used to
purify all intermediates. Chromatography with silica gel (70-230 mesh)
was used to purify substrates. Visualization of UV-inactive materials
was accomplished by using phosphomolybdic acid (PMA) followed
by charring or a combination of I2 vapor followed by a water spray.
(14) Crombie, L.; Heaves, A. D. J. Chem. Soc., Perkin Trans. 1 1992,
1929-1937.
(15) Vogel, A. In Vogel’s Textbook of Practical Organic Chemistry, 4th
ed.; revised by Furniss, B. S., Hannaford, A. J., Rogers, V., Smith, P. W.
G., Tatchell, A. R.; Longman: New York, 1978; pp 387-388.
(16) Rackoff, H. Prog. Lipid Res. 1982, 42, 225-254.
(17) Bradford, M. M. Anal. Biochem. 1976, 72, 248-254.
(18) Garner, P.; Park, J. M.; Malecki, E. J. Org. Chem. 1988, 53, 4395-
4398.
(11) Broadwater, J. A.; Fox, B. G.; Haas, J. A. Fett/Lipid 1998, 100,
103-113.
(12) Lawlor, E. J.; Elson, S. W.; Holland, S.; Cassels, R.; Hodgson, J.
E.; Lloyd, M.; Baldwin, J. E.; Schofield, C. J. Tetrahedron 1994 50, 8737-
8748.
(13) De Jonghe, S.; Van Overmeire, I.; Van Calenbergh, S.; Hendrix,
C.; Busson, R.; De Keukeleire, D.; Herdewijyn, P. Eur. J. Org. Chem. 2000,
3177-3183.
(19) Herold, P. HelV. Chim. Acta 1988, 71, 354-362.
(20) Williams, L.; Zhang, Z.; Shao, F.; Carroll, P. J.; Joullie´, M. M.
Tetrahedron 1996, 52, 11673-11694.
(21) Garner, P.; Park, J. M. J. Org. Chem. 1987, 52, 2361-2364.
(22) Wild, R.; Schmidt, R. R. Liebigs Ann. Chem. 1995, 755-764.

4384 J. Am. Chem. Soc., Vol. 123, No. 19, 2001
SaVile et al.
558 (M+ - CH3), 470 (M+ - (CH3)3Si-O-CH2), 315 [((CH3)3Si-
O-CHCH2CD2(CH2)12CH3)+], 258 [((CH3)3Si-O-CH2CH-NHC-
(O)-(CH2)6CH3)+].
Dihydroceramide Desaturase Assay. The procedure reported by
Michel et al. was followed.4 Rat liver microsomes were prepared as
reported.4 Synthetic D-erythro-sphinganine was acylated with octanoyl
chloride23 and purified by chromatography on silica (CHCl3/MeOH 15:1
v/v) to give the corresponding N-octanoyl-D-erythro-sphinganine 3. The
semitruncated dihydroceramide probe was complexed with BSA as
described for other lipids.24 The solution contained 10 nmol of lipid
substrate, 20 nmol of BSA, 10 µL of EtOH, and 90 µL of phosphate
buffer (100 mM NaH2PO4/Na2HPO4, pH 7.4). A rat liver microsomal
suspension containing 300 µg of protein was added to the substrate/
BSA complex and diluted to a volume of 320 µL with phosphate buffer.
NADH (1 µmol) in 30 µL of phosphate buffer was added to the reaction
mixture and incubated at 37 °C for 30 min.
To reduce mass spectral interferences from endogenous material,
the mixture was cooled to 0 °C on ice and rapidly extracted with 500
µL of hexane. The reaction was terminated with the addition of 500
µL of CHCl3. The phases were separated by centrifugation and the
organic layer was collected. The extraction procedure was repeated
twice with 250 µL of CHCl3. The combined organics were evaporated
under a steady stream of N2 and derivatized with 100 µL of
bis(trimethylsilyl)trifluoracetamide at 25 °C for 1 h. Excess reagent
was evaporated under a stream of N2 and the residue was diluted with
100 µL of CHCl3 for analysis by GC/MS.
Results and Discussion
According to our mechanism (Scheme 1), the first C-H
cleavage step in a desaturase-mediated reaction is energetically
difficult and hence more sensitive to isotopic substitution than
the collapse of the intermediate radical. Thus one way of
determining the site of initial oxidative attack for dihydro-
ceramide ∆4 desaturase is via an unambiguous assessment of
which C-H cleavage is rate-determining. This can be ac-
complished by a measurement of the intermolecular primary
deuterium KIE associated with each C-H bond breaking step
to assess its kinetic importance.25 Our experimental approach
involved incubating a roughly equimolar mixture of the ap-
propriate regiospecifically dideuterated (-CD2-) substrate
analogue and the nondeuterated parent compound with a
convenient source of the ∆4 desaturase. Evaluation of the d0/d1
ratio in the olefinic products by mass spectral examination would
then allow one to compute the competitive KIE isotope effect
for each C-H bond rupture.
Figure 1. Formation of N-octanoyl-D-erythro-sphingosine 4 by rat liver
microsomes as determined by SIM-GC/MS. The traces correspond to
selection of ions at m/z 313(A), 311 (B), and 258 (C). The peaks are
labeled as follows: 3-TMS, trimethylsilyl derivative of 3 (reaction
substrate) and 4-TMS, trimethylsilyl derivative of 4 (reaction product).
The peak marked with an asterisk is the trimethylsilyl derivative of
cholesterol.
experiments (see below), selection of the appropriate diagnostic
ions in the SIM-GC-MS analyses allowed us to make the
measurements of isotopic content with high sensitivity and
accuracy.
The two regiospecifically dideuterated substrates ([4,4-2H2]-
3) and ([5,5-2H2]-3), which were required for this study, were
synthesized de novo by well-precedented procedures involving
Grignard addition to suitably protected L-serinal18,19,20 followed
by chromatographic separation of the threo and erythro adducts
(1:2 ratio) as the t-Boc derivatives. The purified erythro isomers
were deprotected and then acylated with octanoyl chloride to
give ([4,4-2H2]-3) and ([5,5-2H2]-3 in 4% and 5% overall yield,
respectively (Scheme 2). The erythro and threo isomers could
be distinguished on the basis of well-documented differences
To examine the dihydroceramide ∆4 desaturase system with
this method, a reliable GC-MS assay of sufficient sensitivity
had to be used. The procedure used by Michel4 to measure
desaturation of 14C-labeled semitruncated dihydroceramide was
followed for this purpose. In our case, the enzymatic reaction
was monitored by GC-MS analyses of silylated extracts.
Trimethylsilyl derivatives of ceramides give characteristic
fragments in their mass spectra, with diagnostic ions arising
from cleavage of the C2-C3 bond.26a,b For incubations with
nonlabeled N-octanoyl-D-erythro-sphinganine 3, the identity of
both substrate and product was confirmed by mass spectral
analysis of a silylated extract. Diagnostic fragment ions were
m/z 258 and 313 (trimethylsilylated substrate) and m/z 258 and
311 (trimethylsilylated product) (Figure 1). In the competitive
1
in the H NMR of the N-acetyl derivatives.27 GC/MS analysis
of the final deuterated products revealed that each isotopomer
consisted essentially entirely of dideuterated species.
In the competitive KIE experiments, a 1:1 mixture of each
dideuterated substrate with its nondeuterated parent was incu-
bated with the microsomes, containing the dihydroceramide
desaturase, under conditions similar to those described by Michel
et al.4 Care was taken to maintain the level of conversion below
10% to prevent possible enhancement of the d2/d0 ratio in the
substrates.28 The deuterium content of the ∆4 olefinic products
was assessed by GC/MS as described in the Experimental
(23) Kashiwagi, M.; Mikami, T.; Chiba, M.; Chiba, S.; Matsumoto, H.;
Akino, T.; Gasa, S. Biochem. Mol. Biol. Int. 1997, 42, 1071-1080.
(24) Pagano, R. E. Methods Cell Biol. 1989, 29, 75-85.
(25) Klinman, J. P. In Transition States in Biochemical Processes;
Gandour, R. L., Schowen, R. L., Eds.; Plenum Press: New York, 1978; pp
165-200.
(26) (a) Olsson, N. U.; Kaufmann, P.; Dzeletovic, S. J. Chromatogr. B,
Biomed. Sci. Appl. 1997, 698, 1-8. (b) Raith, K.; Darius, J.; Neubart, R.
H. H. J. Chromatogr. A 2000, 876, 229-233.
(27) Bielawska, A.; Crane, H. M.; Liotta, D.; Obeid, L. M.; Hannun, Y.
A. J. Biol. Chem. 1993, 268, 26226-26232.

Dihydroceramide ∆4 Desaturase Initiates Substrate Oxidation
J. Am. Chem. Soc., Vol. 123, No. 19, 2001 4385
Table 1. Intermolecular Isotopic Discrimination in ∆4 Desaturation
of [4,4-2H2]-3 and [5,5-2H2]-3
Scheme 2
isotopic ratio
substrates
products
d0:4d1
8.28
d0:4d2
1.04
d0:5d2
d0:5d1
1.34
KIEa
8.0 ( 0.8
1.02 ( 0.07
1.32
a The average KIE (three different incubations) ( standard deviation.
The stereochemical evidence also points to a common site of
attack since it is the pro-(R) hydrogen at C-4 that is removed
in both enzymatic reactions.29,30 This picture is very strongly
reminiscent of the connection that has been established between
the structurally related castor oleate 12-hydroxylase and mi-
crosomal ∆12 oleate desaturase.8,31,32 By using a KIE approach,
it was shown that the latter enzyme initiated oxidation at the
same carbon that is attacked by the corresponding 12-hydroxy-
lase (KIE, ∆12 oleate desaturase: kH/kD ) 7.3 (C-12); kH/kD )
1.05 (C-13)).8 A similar trend in KIE values has also been
observed in the cytochrome P450-catalyzed 4-desaturation of
the anti-epileptic drug valproic acid (2-propylbutanoic acid)sa
process that is accompanied by the formation of a 4-hydroxy-
lated metabolite as the major product.33
In conclusion, the results presented in this paper have two
important consequences for ceramide research. First, our data
strongly support the use of existing sequence information for
dihydroceramide 4-hydroxylase to probe for corresponding ∆4
desaturase in mammalian systems. Second, knowing that the
site of initial oxidative attack is at C-4 should aid in the design
of and search for inhibitors of this enzyme. Progress at both of
these fronts is crucial to a more complete understanding of
ceramide biosynthesis and its role in apoptotic pathways.
Acknowledgment. This work was supported by N.S.E.R.C.
(Grant OGP-2528 to P. H. B.), Comisio´n Asesora de Investi-
gacio´n Cientifica y Te´cnica (Grant AGF 98-0844), and Comis-
sionat per a Universitats i Recerca from the Generalitat de
Catalunya (Grant 97SGR-0021 to G. F.). We are indebted to
Dr. Josefina Casas for her help in the preparation of microsomes.
We would like to dedicate this paper to Prof. Francisco Camps
on the occasion of his 65th birthday.
Section. Product kinetic isotope effects (kH/kD) were calculated
by using the following ratio: [% d0(product)/% d1(product)]/
[% d0(substrate)/% d2(substrate)]. The d0/d2 ratio of the sub-
strates was determined prior to incubation. Analysis of the
products of ∆4 desaturation 4 revealed a large primary deuterium
isotope (8.0 ( 0.8) for the carbon-hydrogen bond cleavage at
C-4 while the C5-H bond-breaking step was essentially
insensitive to deuterium substitution (KIE ) 1.02 ( 0.07). The
analytical data are displayed in Table 1.
These results provide the first pieces of information on the
mechanism of dihydroceramide ∆4 desaturase. The KIE data
clearly indicate that C-H cleavage at C-4 but not C-5 is
kinetically important. According to our model (Scheme 1), this
implies that initial oxidative attack occurs at C-4 and strengthens
the notion that the production of phytosphingosines via 4-hy-
droxylation and formation of sphingosines via 4,5-desaturation
are closely linked at the mechanistic level (see Introduction).
Supporting Information Available: Mass spectra of 3-TMS
and 4-TMS showing the ions which were used for analytical
purposes (PDF). This material is available free of charge via
the Internet at http://pubs.acs.org.
JA010088W
(28) Walsh, C. In Enzymatic Reaction Mechanisms; W. H. Freeman: San
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