Cationic substrates of soybean lipoxygenase-1

Article abstract of DOI:10.1016/j.bioorg.2010.12.003

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to 13(S)-hydroperoxy-9(Z),11(E)- octadecadienoate. The manner in which SBLO-1 binds substrates is uncertain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion (LTMA) to give primarily13(S)-hydroperoxy-9(Z), 11(E)-octadecadienyltrimethylammonium ion. The rate of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate substrates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.

Full text of DOI:10.1016/j.bioorg.2010.12.003

Bioorganic Chemistry 39 (2011) 94–100
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Cationic substrates of soybean lipoxygenase-1
⇑
Lucas E. Chohany, Kathleen A. Bishop, Hannah Camic, Stephen J. Sup, Peter M. Findeis, Charles H. Clapp
Department of Chemistry, Bucknell University, Lewisburg, PA 17837, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 November 2010
Available online 30 December 2010
Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of 1,4-dienes to produce conjugated diene
hydroperoxides. The best substrates are anions of fatty acids; for example, linoleate is converted to
13(S)-hydroperoxy-9(Z),11(E)-octadecadienoate. The manner in which SBLO-1 binds substrates is uncer-
tain. In the present work, it was found that SBLO-1 will oxygenate linoleyltrimethylammonium ion
(LTMA) to give primarily13(S)-hydroperoxy-9(Z),11(E)-octadecadienyltrimethylammonium ion. The rate
of this process is about the same at pH 7 and pH 9 and is about 30% of the rate observed with linoleate at
pH 9. At pH 7, SBLO-1 oxygenates linoleyldimethylamine (LDMA) to give primarily 13(S)-hydroperoxy-
9(Z),11(E)-octadecadienyldimethylamine. The oxygenation of LDMA occurs at about the same rate as
LTMA at pH 7, but more slowly at pH 9. The results demonstrate that SBLO-1 will readily oxygenate sub-
strates in which the carboxylate of linoleate is replaced with a cationic group, and the products of these
reactions have the same stereo- and regiochemistry as the products obtained from fatty acid substrates.
Ó 2011 Elsevier Inc. All rights reserved.
Keywords:
Lipoxygenase
Specificity
Stereochemistry
Substrate binding
Linoleyldimethylamine
Linoleyltrimethylammonium ion
1. Introduction
cling of the iron between the ferrous and ferric states [15,16].
The iron is located in the center of a large helical domain [17,18].
Lipoxygenases catalyze the formation of hydroperoxides from
the 1,4-diene units of polyunsaturated fatty acids and derivatives
[1,2]. In plants, lipoxygenases are involved in storage-lipid mobili-
zation and in the synthesis of jasmonic acid and other oxylipins [3].
In animals, lipoxygenases catalyze key steps in the synthesis of leu-
kotrienes and other inflammatory mediators [4] and appear to be
involved in membrane modification during cell differentiation
and modification [1,5]. Inhibitors of human lipoxygenases are of
interest as potential drugs for asthma [6], inflammation [4], ath-
erosclerosis [7] and cancer [8].
The most extensively studied lipoxygenase is soybean lipoxyge-
nase-1 (SBLO-1). This enzyme catalyzes the oxygenation of linole-
ate to 13-(S)-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD,
Scheme 1). SBLO-1 will also oxygenate other fatty acids [9], phos-
pholipids [10–12], and various other substrates that have a 1,4-
Crystal structures are available for several other plant lipoxygenas-
es [19,20], a lipoxygenase from coral [21], and one mammalian
lipoxygenase [22,23], and these enzymes have very similar second-
ary and tertiary structures to SBLO-1.
An important unanswered question about SBLO-1 and other lip-
oxygenases is the manner in which substrates bind. Answering this
question is necessary in order to fully understand how these en-
zymes control the stereo- and regiochemistry of oxygenation [24]
and would also aid in the design of lipoxygenase inhibitors. An
additional challenge for SBLO-1 and mammalian 15-lipoxygenase
is to account for the fact that these enzymes will oxygenate not
only free fatty acids but also larger substrates such as phospholip-
ids [10–12,25].
It has been known for some time that SBLO-1 will oxygenate
fatty acid anions more readily than neutral substrates such as
methyl linoleate. In considering possible modes of substrate bind-
ing, we became interested in whether SBLO-1 would oxygenate
substances in which the carboxylate group of linoleate was re-
placed by a positively charged group. In 1977, Bild et al. [13] re-
ported that oxygen uptake was observed when SBLO-1 was
incubated with 10,13-nonadecadienamine and noted that the ami-
no group of this substrate was probably positively charged under
the conditions of their experiments. Interestingly, the rate of oxy-
gen uptake was higher than observed with most neutral substrates.
The interpretation of this result is uncertain, because the products
of the reaction were not identified. In this paper, we report the
synthesis of linoleyldimethylamine (LDMA, Scheme 2) and
diene unit that begins in the
x6 position of an alkyl chain
[13,14]. Like almost all lipoxygenases, SBLO-1 is a non-heme iron
protein, and its catalytic mechanism appears to involve redox cy-
Abbreviations: BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; EIMS, electron-impact mass spectrometry; ESI-
MS, electrospray ionization mass spectrometry; GC/MS, gas chromatography/mass
spectrometry; 13-HOD, 13(S)-hydroxy-9(Z),11(E)-octadecadienoate; 13-HPOD, 13-
(S)-hydroperoxy-9(Z),11(E)-octadecadienoate;
LDMA,
linoleyldimethylamine;
LTMA, linoleyltrimethylammonium ion; NMR, nuclear magnetic resonance; SBLO-
1, soybean lipoxygenase-1; TLC, thin layer chromatography.
⇑
Corresponding author. Fax: +1 570 577 1739.
E-mail address: cclapp@bucknell.edu (C.H. Clapp).
0045-2068/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2010.12.003

L.E. Chohany et al. / Bioorganic Chemistry 39 (2011) 94–100
95
Scheme 1. Reaction catalyzed by SBLO-1.
tate/acetic acid (55:45:2). This procedure yields the acetate salt of
LDMA, which can be converted to the free base by treatment with
5 M NaOH followed by extraction with dichloromethane and con-
centration to an oil. 1H NMR (400 MHz, CDCl3): d 5.36 (4H, m), 2.77
(2H, t, J = 6.5 Hz), 2.25 (2H, m), 2.22 (6H, s), 2.05 (4H, q, J = 6.7 Hz),
1.45 (2H, m), 1.29 (16H, m), 0.89 (3H, t, J = 6.8 Hz). EIMS: m/z 293
(M+).
2.1.2. Linoleyltrimethylammonium iodide (LTMA)
A
solution of 275 mg (0.94 mmol) of LDMA and 773 mg
(6.7 mmol) of methyl iodide in 15 mL of acetone was stirred for
20 h at room temperature. Concentration in vacuo yielded an amor-
phous gum. 1H NMR (400 MHz, CDCl3) d 5.36 (4H, m), 3.60 (2H, m),
3.47 (9H, s), 2.77 (2H, t, J = 6.3 Hz), 2.04 (4H, q, J = 6.7 Hz), 1.77 (2H,
m), 1.26 (16H, m), 0.89 (3H, t, J = 6.5 Hz). 13C NMR (75 MHz,
CDCl3): d 130.5, 130.2, 128.3, 128.1, 67.5, 54.0, 31.7, 29.8, 29.6,
29.5, 29.4, 29.3, 27.4, 27.4, 26.3, 25.7, 23.4, 22.8, 14.3. ESI-MS:
m/z 309 (M+). TLC [CHCl3/MeOH/H2O (64:25:4)] Rf = 0.32.
Scheme 2. Synthesis of LDMA and LTMA.
2.2. Enzymes and assays
SBLO-1 was purified from soybeans by the procedure of Axelrod
linoleyltrimethylammonium iodide (LTMA) and demonstrate that
these substances undergo oxygenation by SBLO-1 to give products
analogous to those obtained with fatty acid substrates.
et al. [27], and its concentration was determined spectrophotomet-
0:1
280nm
rically using A
¼ 1:6 [28]. One unit of lipoxygenase activity is
the amount of enzyme that will convert linoleic acid to 13-HPOD at
a rate of 1.0
[29].
lmole/min under the conditions described previously
2. Materials and methods
Oxygen consumption was measured polarographically with a
Clarke-type electrode in a Hansatech D.W. Oxygen Electrode Unit
maintained at 25 °C with a circulating water bath. Reactions were
initiated by addition of enzyme (2.5–10 lL) to solutions of sub-
strate in either 50 mM potassium phosphate, pH 7.0, or 50 mM bo-
2.1. Synthesis
The synthesis of LDMA and LTMA is outlined in Scheme 2, and
the procedures are described below. Methyl linoleate (Sigma/
Aldrich) was reduced to linoleyl alcohol (1) by a published method
[26]. NMR spectra were obtained on a Varian 400 MHz spectrome-
ter or a Bruker ARX 300 MHz instrument, and chemical shifts are
reported relative to tetramethylsilane.
rate, pH 9.0, in a total volume of 0.50 mL.
Peroxide was determined by the ferrous/xylenol orange method
[30] or by using a coupled enzyme assay with glutathione peroxi-
dase and glutathione reductase [31], which were obtained from
Sigma/Aldrich.
2.1.1. Linoleyldimethylamine (LDMA)
Methanesulfonyl chloride (7.1 mmol, 0.81 g) was added drop-
wise at 0 °C to a stirred solution of 1.49 g (5.6 mmol) of linoleyl
alcohol (1) and 0.85 g (8.4 mmol) of triethylamine. The mixture
was stirred for 45 min at 0 °C and 45 min at room temperature.
The reaction mixture was washed with 20-mL portions of water,
10% HCl, 10% NaHCO3, and 10% NaCl, and the organic layer was
concentrated to an oil, which gave a single spot by TLC (CHCl3/
MeOH/H2O (64:25:4) Rf = 0.86) and had a 1H NMR spectrum con-
sistent with the methanesulfonate of linoleyl alcohol: (400 MHz,
CDCl3) d 5.35 (4H, m), 4.22 (2H, t, J = 6.6 Hz), 3.00 (3H, s), 2.77
(2H, t, J = 6.4 Hz), 2.05 (4H, q, J = 7 Hz), 1.75 (2H, quintet,
J = 7 Hz), 1.30 (16H, m), 0.89 (3H, t, J = 6.9 Hz). A solution of
0.94 g (2.7 mmol) of the methanesulfonate, 7.23 g (88.7 mmol) of
dimethylamine hydrochloride and 1.00 g (6.6 mmol) of 1,8-diaza-
bicyclo[5.4.0] undec-7-ene (DBU) in 12 mL of methanol was heated
in a pressure tube at 56 °C for 48 h. The methanol was removed on
a rotary evaporator, and the residue was partitioned between
70 mL of ether and 70 mL of 5% K2CO3. The organic layer was
washed with two 25-mL portions of 1 M NaCl, dried with MgSO4,
and concentrated on a rotary evaporator. LDMA can be purified
by flash chromatography on silica gel 60 with methanol/ethyl ace-
2.3. Mass spectrometry
GC/MS analyses were carried out on a Hewlett–Packard GCD
instrument with a 12 m ꢀ 0.3 mm HPI capillary column. The col-
umn temperature was maintained at 50 °C for 3 min and then in-
creased from 50 to 250 °C at 20o/min. Electrospray MS and MS/
MS experiments were carried out on a Sciex API III+ triple-quadru-
pole instrument operated in the positive ion mode at an ionization
voltage of 3 kV. Samples were infused into the capillary in 3 mM
ammonium formate in methanol/water (1:1). MS/MS experiments
were carried out with argon/nitrogen (9:1) as collision gas and a
collision energy of 30 eV.
2.4. Lipoxygenase-catalyzed oxygenation of LDMA
A 200-mL solution of 0.18 mM LDMA in 50 mM phosphate buf-
fer, pH 7.0, was treated with 20 units of SBLO-1, and the mixture
was stirred vigorously at room temperature and monitored by
measuring the A234 of 1:10 dilutions of aliquots withdrawn period-
ically. After 30 min, the A234 leveled off (at 0.26 in a 1:10 dilution),

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L.E. Chohany et al. / Bioorganic Chemistry 39 (2011) 94–100
Scheme 3. Hydroperoxides and alcohols derived from LDMA and LTMA.
and the reaction mixture was extracted with ether (3 ꢀ 60 mL).
Concentration of the extracts yielded an oil, which was dissolved
in 3 mL of ethanol and treated with 26 mg of NaBH4 at 0 °C. After
30 min of stirring, the excess NaBH4 was destroyed by cautious
addition of acetic acid (ca 1 mL), and the mixture was extracted
with ether (3 ꢀ 10 mL). Concentration of the extracts yielded an
oil, which will be referred to as the reduced product from LDMA.
This material gave a single spot (Rf = 0.26) by TLC in hexanes/meth-
anol (98:2) and a 1H NMR spectrum that contained the resonances
expected for either 2b or 3b (Scheme 3): (300 MHz, CDCl3): d 6.49
(1H, dd, J = 15.1, 11.1 Hz), 5.97 (1H, t, J = 10.9 Hz), 5.67 (1H, dd,
J = 15.1, 6.8 Hz), 5.42 (1H, dt, J = 10.8, 7.6 Hz), 4.15 (1H, q,
J = 6.6 Hz), 2.53 (2H, m), 2.44 (6H, s), 2.18 (2H, q, J = 7.2 Hz).
Approximately 1 mg of this substance in 1.0 mL of methanol was
stirred under H2 for 1 h with 10 mg of 5% Pd on CaCO3. The catalyst
was removed by centrifugation, and the methanol was evaporated
under a stream of nitrogen. The residue was incubated with 10
lL
of pyridine and 10 L of BSTFA for 1 h at room temp. to convert the
l
hydroxyl group to its trimethylsilyl derivative for GC/MS analysis.
2.5. Methylation of reduced product from LDMA
A 3.5-mg portion of the reduced product from LDMA was stirred
with 15 mg of methyl iodide in 4 mL of acetone for 8 h at room
temperature. An additional 40 mg of methyl iodide was added,
and the mixture was stirred for another 16 h. At this point, TLC
(CHCl3/CH3OH/H2O, 65:25:4) indicated complete conversion to a
product with Rf = 0.66. This material was used for the MS/MS spec-
trum in Fig. 4B.
Scheme 4. Stereochemical analysis.
2.6. Lipoxygenase-catalyzed oxygenation of LTMA
To a stirred solution of 20 mg of LTMA in 150 mL of 25 mM
(NH4)2CO3, pH 9.0, was added 6 units SBLO-1. After 35 min the
A234 in a 1:10 dilution had risen to 0.74. The reaction mixture
was concentrated on a rotary evaporator with a mechanical vac-
uum pump. The residue was dissolved in CDCl3, and residual buffer
salts were removed by centrifugation. The 1H NMR spectrum
(400 MHz) contained the signals expected for 4a and/or 5a: d
6.42 (1H, dd, J = 15.2, 11.1 Hz), 5.91 (1H, t, J = 11.0 Hz), 5.60 (1H,
dd J = 15.1, 6.7 Hz), 5.36 (1H, dt, J = 10.7, 7.6 Hz), 4.10 (q,
J = 6.5 Hz), 3.51 (m), 3.31 (s). Signals for unreacted LTMA were also
present. About 5 mg of this material in 1.0 mL of ethanol was trea-
ted with NaBH4 (10 mg), and the mixture was stirred for 30 min at
0 °C and 30 min at room temperature. The NaBH4 was quenched
with glacial acetic acid (ꢁ100
lL), and the reaction mixture was
loaded onto a 20 ꢀ 1 cm column of Al2O3. The column was eluted
with ethanol, and the portion of the eluent that absorbed at
234 nm was concentrated. The residue gave an NMR spectrum
similar to that obtained before NaBH4 treatment. This material
was also analyzed by electrospray MS and MS/MS (Fig. 4A).
Fig. 1. Initial rates of oxygenation of linoleic acid, LDMA and LTMA by SBLO-1 at pH
7.0, 25 °C. Each reaction contained 72 lM of the indicated substrate. The slopes are
indicated next to trend lines.

L.E. Chohany et al. / Bioorganic Chemistry 39 (2011) 94–100
97
room temp, the reaction mixture was concentrated under N2 and
the product was purified by preparative TLC (hexanes/ethyl ace-
tate/acetic acid 7:3:1; Rf = 0.60). This material was dissolved in
300 lL of acetic acid and treated with 22 mg of solid KMnO4. The
mixture was incubated for 2 h at 37 °C and then diluted with
1 mL of water and extracted with ether (2 ꢀ 0.5 mL). The ether ex-
tracts were washed with water (1.0 mL) and concentrated to dry-
ness. The residue was methylated with diazomethane to give 7
and/or 8. A synthetic mixture of 7 and 8 was prepared from 6
and racemic methyl 2-hydroxyheptanoate, which was prepared
by a published method [32]. Stereochemical analysis of the product
from LTMA was carried out in the same manner, except that the
TLC purification of the product from the first step was omitted.
3. Results
Fig. 1 shows the rates of O2 consumption that occurred when
increasing concentrations of SBLO-1 were incubated at 25 °C with
72 lM solutions of LDMA, LTMA or linoleic acid in 50 mM potas-
Fig. 2. Initial rates of oxygenation of linoleic acid, LTMA and LDMA by SBLO-1 at pH
9.0, 25 °C. Each reaction contained 72 lM of the indicated substrate. The slopes are
indicated next to trend lines.
sium phosphate buffer, pH 7.0. The data indicate that LDMA and
LTMA undergo enzymatic oxygenation at about equal rates, which
are about 65% of the rate observed with linoleic acid. Fig. 2 shows
the results of a similar experiment, with the same concentration of
substrates, carried out in 50 mM borate buffer, pH 9.0. Linoleic acid
is a considerably better substrate for SBLO-1 at pH 9.0 than at pH
7.0 [13,33], and this gives rise to a 2.4-fold increase in the slope
in the plot of rate vs enzyme when linoleic acid is the substrate.
(Numerical values for the slopes are displayed next to the trend
2.7. Stereochemical analysis
Six milligrams of the reduced product from LDMA was treated
with 59
l
L of pyridine, 18
l
L of methylene chloride and 6
chloride
l
L of
(6,
(S)- -methoxy-
a
a-(trifluoromethyl)phenylacetyl
Scheme 4), which was obtained from Sigma/Aldrich. After 5 h at
Fig. 3. Electron-impact mass spectra of derivatized products from LDMA. (A) Major product. (B) Minor product.

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L.E. Chohany et al. / Bioorganic Chemistry 39 (2011) 94–100
Fig. 4. Electrospray MS/MS spectra of products. (A) Reduced product from LTMA. (B) Methylated reduced product from LDMA. (C) Tentative peak assignments.
lines in the Figures. The slopes cannot be compared visually, since
the scales are different). When LTMA is substrate, the slopes are
identical within error in Figs. 1 and 2, which indicates that this
substrate is equally active at pH 7.0 and 9.0. With LDMA as sub-
strate, oxygenation is barely detectable at pH 9.0.
is characteristic of the conjugated diene in lipoxygenase products.
Assay with glutathione peroxidase [31] indicated that a peroxide
had formed. To establish the structure of the product(s), the reac-
tion was carried out on larger scale, and the products were treated
with NaBH4 to reduce hydroperoxides (2a and/or 3a, Scheme 3) to
the corresponding alcohols (2b and/or 3b). The 1H NMR spectrum
of the resulting material was identical in the d 4–7 region to the
The oxygenation of LDMA catalyzed by SBLO-1 at pH 7.0 gives
rise to a species with an absorption maximum at 234 nm, which

L.E. Chohany et al. / Bioorganic Chemistry 39 (2011) 94–100
99
spectrum of 13-(S)-hydroxy-9(Z),11(E)-octadecadienoate (13-
HOD), which indicates that that the product is a cis–trans diene
with a hydroxyl group on the carbon next to the trans double bond.
The spectrum is consistent with either 2b or 3b. To distinguish be-
tween these regioisomers, the product mixture was catalytically
hydrogenated, trimethylsilylated and analyzed by GC/MS. The
mass spectrum of the major product (Fig. 3A) was consistent with
the presence of a trimethylsilyloxy group on carbon-13. Also de-
tected was a minor product (about 3%) with a mass spectrum con-
sistent with the trimethylsilyloxy group on carbon-9 (Fig. 3B).
These results indicate that the action SBLO-1 on LDMA produces
a mixture of the 13-hydroperoxide, 2a, and the 9-hydroperoxide,
3a, in a ratio of 97:3.
barely detectable. Based on the reasonable assumption that the
protonated form of LDMA should behave similarly to LTMA, the
lower activity of LDMA at pH 9.0 is likely due to a lower concentra-
tion of protonated LDMA at pH 9.0 than at pH 7.0. Owing to the
very limited solubility of LDMA in aqueous solution, its pKa cannot
be reliably determined. Smaller tertiary amines have pKa values of
about 10 [37]. As noted below, LDMA appears to aggregate under
the conditions of our experiments, and this would probably per-
turb the ionization equilibrium in favor of the neutral species
and shift the apparent pKa to a lower value [38]. The pKa of N-octa-
decylamine in neutral or cationic micelles has been found to be
8.5–9.0, compared with values of about 10.6 for shorter chain pri-
mary aliphatic amines in aqueous solution [39]. Aggregation of lin-
oleic acid gives rise to apparent pKa values of 7–8 [13,33], which
indicate a sizeable perturbation in favor of the neutral species.
Solutions of LTMA and LDMA at micromolar concentrations at
pH 7.0 and pH 9.0 are slightly opaque, indicating that these sub-
stances are not completely soluble at these concentrations. For this
reason, we carried out the kinetic studies using O2 uptake rather
than spectroscopic methods. We have not examined in detail the
dependence of the rates on substrate concentration, since the ac-
tual concentration of dissolved substrate is unlikely to be equal
to the nominal concentration. The unprotonated form of LDMA is
likely to be less soluble than the protonated form, and this may
contribute to the low activity of LDMA at pH 9.0.
The stereochemistry of the major product was determined by a
modification of procedures developed by Hamberg [34] and Gard-
ner [35]. The NaBH4-reduced product was derivatized with (S)-
a-
methoxy- -(trifluoromethyl)phenylacetyl chloride (6) [36], and
a
the resulting ester was degraded as shown in Scheme 4. This pro-
cedure should convert the S enantiomer of 2b to 7 and the R enan-
tiomer of 2b to 8. GC/MS analysis of the degradation products
under the conditions described in Materials and Methods gave
peaks at 11.4 min and 11.2 min in a ratio of 90:10. The retention
times and mass spectra were identical to those obtained on a
synthetic mixture of 7 and 8 produced by reaction of (S)-
a-meth-
oxy- -(trifluoromethyl)phenylacetyl chloride with racemic methyl
a
2-hydroxyheptanoate. The degradation shown in Scheme 4 was
also carried out on (S)-13-HOD, and in this case, the relative inten-
sities of the peaks at 11.4 min and 11.2 min were 91:09. This result
establishes that the diasteriomer eluting at 11.4 min is 7. Conse-
quently, the results obtained on the products from LDMA indicate
that the stereochemistry of 2a at carbon-13 is predominantly S.
The oxygenation of LTMA by SBLO-1 at pH 9.0 also gave rise to a
peroxide, as shown by the ferrous/xylenol orange assay [30]. Since
initial studies indicated that the product could not be extracted
from aqueous solution, a volatile buffer, (NH4)2CO3, was employed
for preparative experiments and removed in vacuo. The residue
gave an NMR spectrum that contained the expected signals for
either 4a or 5a. Following reduction by NaBH4, electrospray MS
gave a peak at m/z 325, the expected M + ion for either 4b or 5b.
The MS/MS spectrum obtained by collision-induced fragmentation
of the m/z 325 ion is shown in the in Fig. 4A. To facilitate the inter-
pretation of this spectrum, the 97:3 mixture of 2b and 3b obtained
in the experiments with LDMA was methylated with CH3I to pro-
vide a sample that should be a 97:3 mixture of 4b and 5b. The elec-
trospray MS/MS spectrum of this material (Fig. 4B) is virtually
identical to the spectrum in Fig. 4A. The close correspondence of
these spectra indicates that oxygenation of LTMA occurs primarily
at carbon-13 to give 4a. Tentative assignments of some of the
prominent fragments in the MS/MS spectra are given in Fig. 4C.
Degradation of the product from LTMA according to Scheme 4 gave
7 and 8 in a ratio of 80:20. This result indicates that the major
product has predominantly the S configuration at carbon-13.
It is now clear that SBLO-1 will oxygenate substrates in which
the polar end is either negative, positive or zwitterionic, as in the
case of phospholipids. Uncharged substrates can also be oxygen-
ated [13,40], especially if their solubility is enhanced with deter-
gents. For example, diacylglycerides that contain linoleoyl side
chains are good substrates in the presence of deoxycholate [40].
The broad specificity of SBLO-1 with respect to the polar end of
its substrates is consistent with proposals that interaction of the
protein with the alkyl terminus is the primary determinant of
specificity [9,41].
As noted in the Introduction, the manner in which SBLO-1 binds
substrates is uncertain. One possibility is that linoleate and other
fatty acid substrates bind in a manner that is similar to the binding
of 13-HPOD to SBLO-3, with the carboxylate of the fatty acid close
to arginine-707 [19]. Docking and site-directed mutagenesis
experiments provide some support for this proposal [42]. With this
sort of model for substrate binding, one might expect that replace-
ment of the negatively charged carboxylate with a positively
charged group would greatly lower substrate activity owing to
an unfavorable interaction with arginine-707. An alternative possi-
bility is that the carboxylate of linoleate binds at or near the sur-
face of the protein, and several proposals of this nature have
been put forth [2,21,24,43]. Binding in this manner can explain
the activity of SBLO-1 with larger substrates such as phospholipids.
If the carboxylate group of linoleate binds near the protein surface,
it could be stabilized either by interaction with a positively
charged residue or by interaction with water. In the latter case,
one would expect that replacing the carboxylate group of linoleate
with a positively charged group might not result in a drastic reduc-
tion in substrate activity, since interaction with water is favorable
with either a positive or negative charge. The robust activity that
we observe with LTMA and LDMA is consistent with this sort of
model.
4. Discussion
The results with LTMA unequivocally demonstrate that SBLO-1
will oxygenate a substrate in which the carboxylate group of lino-
leic acid is replaced with a positively charged group. The activity is
about the same at pH 9.0 and pH 7.0. The major product at pH 9.0
was identified as 13(S)-hydroperoxy-9(Z),11(E)-octadecadienyl-
trimethylammonium ion (4a). Thus, the reaction proceeds with
the same regio- and stereochemistry as occurs with linoleate.
At pH 7.0, LDMA is oxygenated at about the same rate as LTMA,
and the major product is 13(S)-hydroperoxy-9(Z),11(E)-octadeca-
dienyldimethylamine (2a). At pH 9.0, the oxygenation of LDMA is
At pH 7.0, the reaction catalyzed by SBLO-1 on LDMA shows a
very high level of regiospecificity in favor of oxygenation at car-
bon-13. Only about 3% of the 9-hydroperoxide is formed. In con-
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about 20% of 9-HPOD at pH 7, compared with about 5% at pH 9.0
[44]. The higher yield of 9-HPOD formed at pH 7.0 has been attrib-
uted to ‘‘reverse binding’’ in which neutral linoleic acid is proposed
to bind with the protonated carboxyl group occupying the site

100
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Acknowledgments
We are grateful to Karen Vasey for the synthesis of racemic
methyl 2-hydroxyheptanoate. This work was supported by the Ste-
phen G. Hobar Memorial Research Fund and the Bucknell Program
for Undergraduate Research. The electrospray mass spectrometer
used in this work was a gift from the Department of Drug Metab-
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