N-linoleoylamino acids as chiral probes of substrate binding by soybean lipoxygenase-1

Article abstract of DOI:10.1016/j.bioorg.2018.03.010

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein. To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution should show little or no sensitivity to the stereochemical configuration of the amino acid moiety. Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both good substrates with k_cat/K_m values that are equal within error and about 40% higher than k_cat/K_m for linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT) were complicated by the low critical micelle concentrations (CMC = 6–8 μM) of these substances. Below the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products is not appreciably affected by the configuration of the amino acid moiety. The absence of stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT, and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts with it only weakly.

Full text of DOI:10.1016/j.bioorg.2018.03.010

Accepted Manuscript
N-Linoleoylamino Acids as Chiral Probes of Substrate Binding by Soybean
Lipoxygenase-1
Charles H. Clapp, Justin Pachuski, Natasha F. Bassett, Kathleen A. Bishop,
Gillian Carter, Megan Young, Thomas Young, Yuhan Fu
PII:
S0045-2068(17)30983-5
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.010
YBIOO 2300
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
6 January 2018
25 February 2018
7 March 2018
Please cite this article as: C.H. Clapp, J. Pachuski, N.F. Bassett, K.A. Bishop, G. Carter, M. Young, T. Young, Y.
Fu, N-Linoleoylamino Acids as Chiral Probes of Substrate Binding by Soybean Lipoxygenase-1, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.03.010
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N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
Charles H. Clapp*, Justin Pachuski, Natasha F. Bassett, Kathleen A. Bishop,
Gillian Carter, Megan Young, Thomas Young, and Yuhan Fu.
Department of Chemistry, Bucknell University, Lewisburg, PA. 17837
*Corresponding author: cclapp@bucknell.edu
Declarations of interest: none

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
Highlights
 Soybean lipoxygenase (SBLO-1) oxygenates N-linoleoyl-L-valine (LLV), N-linoleoyl-D-valine
(LDV) N-linoleoyl-D-tryptophan (LDT), and N-linoleoyl-L-tryptophan (LLT) primarily at carbon-13
of the linoleoyl moiety.
 Values of kcat/Km for LLV and LDV are identical within error.
 LDT is a better substrate than LLT by about a factor of 3.
 The absence of stereoselectivity with LLV and LDV and the modest magnitude of the
stereoselectivity with LLT and LDT suggest that the amino acid moiety interacts only weakly with
the protein.
Abbreviations: BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CMC, critical micelle concentration;
ENDOR, electron-nuclear double resonance; EPR, electron paramagnetic resonance; ESI, electrospray
ionization; GC/MS, gas chromatography/mass spectrometry; HPLC, high pressure liquid
chromatography; 9-HODE, 9-hydroxy-10(E),12(Z)-octadecadienoic acid; 13-HODE, 13-hydroxy-
9(Z),11(E)-octadecadienoic acid; 13-HPOD, 13(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid; HRMS,
high resolution mass spectrometry; LDT, N-linoleoyl-D-tryptophan; LLT, N-linoleoyl-L-tryptophan;
LDV, N-linoleoyl-D-valine; LLV, N-linoleoyl-L-valine; MS/MS, tandem mass spectrometry; NMR,
nuclear magnetic resonance; SBLO-1, soybean lipoxygenase-1.
2

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
Abstract
Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to
produce conjugated diene hydroperoxides. Soybean lipoxygenase-1 (SBLO-1) has been the subject of
intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is
uncertain. Previous studies suggest that the fatty acyl group of the substrate binds in an internal cavity
near the catalytic iron with the polar end at the surface of the protein or perhaps external to the protein.
To test this model, we have investigated two pairs of enantiomeric N-linoleoylamino acids as substrates
for SBLO-1. If the amino acid moiety binds external to the protein, the kinetics and product distribution
should show little or no sensitivity to the stereochemical configuration of the amino acid moiety.
Consistent with this expectation, N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV) are both
good substrates with kcat/Km values that are equal within error and about 40% higher than kcat/Km for
linoleic acid. Experiments with N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT)
were complicated by the low critical micelle concentrations (CMC = 6–8 M) of these substances. Below
the CMC, LDT is a better substrate by a factor of 2.7. The rates of oxygenation of LDT and LLT
continue to rise above the CMC, with modest stereoselectivity in favor of the D enantiomer. With all of
the substrates tested, the major product is the 13(S)-hydroperoxide, and the distribution of minor products
is not appreciably affected by the configuration of the amino acid moiety. The absence of
stereoselectivity with LLV and LDV, the modest magnitude of the stereoselectivity with LLT and LDT,
and the ability micellar forms of LLT and LDT to increase the concentration of available substrate are all
consistent with the hypothesis that the amino acid moiety binds largely external to SBLO-1 and interacts
with it only weakly.
Key Words: Lipoxygenase, specificity, stereoselectivity, substrate binding, N-linoleoylamino acids.
3

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
1. Introduction
Lipoxygenases catalyze the oxygenation of Z,Z-1,4-diene units in polyunsaturated fatty acids and
their derivatives to produce conjugated diene hydroperoxides [1,2]. These enzymes are widespread in
plants [3] and animals [4], and they also occur in prokaryotes [5]. Lipoxygenases contribute to the
synthesis of important signaling molecules, including jasmonic acid [6] in plants and leukotrienes,
lipoxins and resolvins in animals [7–9]. Some lipoxygenases act on phospholipids [10–13] and appear to
be involved in membrane modification [1,14], storage-lipid mobilization [15], and ferroptosis [16].
Inhibitors of human lipoxygenases are of interest as potential drugs for the treatment of inflammation,
asthma, cancer and other disorders [4,7,8,17,18].
Much of our mechanistic insight into lipoxygenases comes from studies on soybean lipoxygenase-1
(SBLO-1). This enzyme catalyzes the oxygenation of linoleate to primarily 13(S)-hydroperoxy-
9(Z),11(E)-octadecadienoate (13-HPOD) [19]. SBLO-1 will also oxygenate other fatty acids,
phospholipids and fatty acid derivatives that have a Z,Z 1,4-diene unit in the 6 position, and the major
product contains oxygen at that position [10–13, 20]. Like most lipoxygenases, SBLO-1 is a nonheme
iron protein [21]. The catalytic mechanism (Scheme 1) is thought to involve transfer of a hydrogen atom
from the bisallylic carbon of the substrate to a ferric hydroxide species in a process that involves
hydrogen tunneling [21–24]. The resulting pentadienyl radical reacts with O2, with the enzyme likely
providing an oxygen channel to control the regio- and stereochemistry [25]. The mechanism postulates
that the bisallylic carbon of the substrate binds near the Fe+3 ion, and this hypothesis is strongly supported
by recent ENDOR studies using samples of
linoleate labelled with 13C and SBLO-1 in
which the catalytic iron has been replaced
by manganese [26].
The iron in SBLO-1 is located in the
center of a large helical domain [27, 28].
4

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
The manner in which substrates bind to SBLO-1 and other lipoxygenases in order to position the bisallylic
carbon close to the iron has been difficult to establish, and progress towards this goal has been reviewed
[29, 30]. The ability of SBLO-1 to oxygenate phospholipids and other substrates with large substituents at
the polar terminus is most readily accommodated by the hypothesis that substrates bind with the alkyl tail
in an internal cavity close to the iron and the polar terminus near the surface [1, 31]. Binding in this
manner would allow large substituents at the polar terminus to remain largely outside the protein. This
binding model is supported by recent studies with spin-labelled substrate analogues [29, 32]. The model
requires some reorganization of the surface of the protein in order to provide access to the internal cavity,
and recent studies suggest that the mobility of helix-2 plays a role in providing this access [33].
Our group has reported that SBLO-1 will oxygenate linoleyltrimethylammonium ion, in which the
negatively charged carboxylate group of linoleate is replaced by a positively charged quaternary
ammonium group [34]. This finding is consistent with the hypothesis that the polar terminus of substrates
has minimal interaction with the protein. As a further test of this model, we have investigated the action
of SBLO-1 on the N-linoleoylamino acids shown in Scheme 2. In each case, the major product was
determined to be the 13(S)-hydroperoxide (Scheme 2); as in the case of linoleate [19], small amounts of
other isomeric products were detected. Our goal in these studies was to determine whether the
stereochemical configuration of the amino acid moiety
affects the kinetics and product distribution. If the amino
acid moiety binds external to the protein, the activity of
SBLO-1 on these substances is expected to show little or
no sensitivity to the configuration of the chiral center.
There has been a previous report of activity of SBLO-1 on
N-arachidonoylamino acids [35], but the work reported
here is the first to focus on the stereoselectivity of the
action of SBLO-1 on substrates of this kind.
5

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
2. Materials and Methods
2.1. Materials
SBLO-1 was purified by the method of Axelrod [36], and its concentration was determined using
A0.1280nm = 1.6 [37]. Linoleoyl chloride, methyl L-valine hydrochloride, methyl D-valine hydrochloride,
methyl L-tryptophan hydrochloride, and methyl D-tryptophan hydrochloride were obtained from Sigma
Aldrich. 13(S)-HPOD was produced enzymatically from linoleic acid using SBLO-1 and reduced with
NaBH4 to give 13(S)-hydroxy-9(Z),11(E)-octadecadienoic acid (13(S)-HODE). 9(S)-HODE, (±)13-
HODE, and (±)9-HODE were obtained from Cayman Chemical. Silica gel 60A for flash chromatography
was obtained from VWR Scientific.
2.2. Synthesis of N-linoleoyl-L-valine (LLV) and N-linoleoyl-D-valine (LDV)
A solution of 1.00 g (3.35 mmol) of linoleoyl chloride in 5 mL of chloroform was added at room
temperature to a stirred mixture of 0.61 g (3.6 mmol) of methyl L-valine hydrochloride and 0.56 g (4.1
mmol) of potassium carbonate in 5.0 mL of chloroform and 5.0 mL of deionized water. The mixture was
stirred for 1.25 h. The chloroform layer was separated, and the aqueous layer was extracted with two 10-
mL portions of chloroform. The combined chloroform solutions were dried with anhydrous magnesium
sulfate and concentrated by rotary evaporation to a colorless oil, which was purified by flash
chromatography [38] on a 30 × 3.5 cm column of silica gel using hexanes/ethyl acetate (9:2) to yield an
oil with an NMR spectrum consistent with the methyl ester of N-linoleoyl-L-valine. The methyl ester was
hydrolyzed by stirring overnight at room temperature with a mixture of 15 mL of 1.0 M aqueous KOH
and 15 mL of methanol. The mixture was acidified and extracted with ethyl acetate (3 × 15 mL). The
combined ethyl acetate extracts were dried (anhyd. MgSO4) and concentrated to a clear oil. Purification
by flash chromatography using hexanes/ethyl acetate/ acetic acid (6:4:0.2) gave a colorless oil with
spectroscopic properties consistent with N-linoleoyl-L-valine: 1H NMR (400 MHz, CDCl3)  6.02 (d, J =
8.4 Hz, 1H), 5.35 (m, 4H), 4.58 (dd, J=8.4 Hz, 4.7 Hz), 2.77 (t, J = 6.0 Hz, 2H), 2.26 (t, J = 7.3 Hz, 2H),
2.23 (m, 1H), 2.04 (m, 4H), 1.64 (t, J = 7 Hz, 2H), 1.3 (m, 14H), 0.99 (d, J = 6.8 Hz, 3H), 0.95 (d, J = 7.0
6

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
Hz, 3H), 0.89 (t, J = 6.4 Hz, 3H). 13C NMR (100 Mz, CDCl3) δ: 175.5, 173.9, 130.2, 130.0, 128.1, 127.9,
57.0, 36.7, 31.5, 30.9, 29.6, 29.3, 29.21, 29.18, 29.12, 27.19, 27.18, 25.67, 25.62, 22.6, 19.0, 17.7, 14.0.
HRMS (ESI, orbitrap) m/z: [M-H]–: Calcd for C23H40O3N 378.3014; Found 378.3012.
N-Linoleoyl-D-valine was synthesized by the same method used for LLV and gave identical
spectroscopic properties. To assess the stereochemical purity of LLV and LDV, a small sample of each
was hydrolyzed, and the resulting valine was derivatized with o-phthaldialdehyde and N-acetyl-L-
cysteine, according to the method of Aswad [39]. This derivatization gives different diastereomers
(depending on the configuration of the valine), which can be separated by HPLC and detected by
fluorescence. Both samples were found to have stereochemical purities of greater than 97% (Figure S1).
2.3. Nonenzymatic Autoxidation of LLV methyl ester
A mixture of the methyl ester of LLV (53 mg, 0.13 mmol) and -tocopherol (2.6 mg) was
incubated at 40oC and periodically flushed with oxygen [40]. After 96 h, the reaction mixture was
dissolved in 11.4 mL of ethanol, cooled on ice, and treated with 93 mg of sodium borohydride, which was
added in several portions over 5 min with stirring. After 30 min of stirring at 0oC and 30 min at room
temperature, the reaction mixture was cooled on ice and treated with acetic acid until gas evolution
ceased. Water (13 mL) was added, and the mixture was extracted with diethyl ether (3 × 20 mL). The
combined ether extracts were concentrated to a yellow oil that was purified by preparative layer
chromatography on a silica gel plate (20 × 20 × 0.1 cm) with fluorescent indicator. The uv-absorbing
component at Rf=0.36 was eluted from the plate with ethyl acetate. Evaporation of the ethyl acetate gave
an oil, which gave four components on normal-phase HPLC (Figure 4C), two of which have NMR
spectra consistent with E,E isomers. The appearance of these isomers was unexpected, since the presence
of -tocopherol eliminates the formation of E,E isomers with methyl linoleate [40].
2.4. Synthesis of N-linoleoyl-L-tryptophan (LLT) and N-linoleoyl-D-tryptophan (LDT)
These substrates were synthesized from linoleoyl chloride and methyl-L-tryptophan hydrochloride
or methyl-D-tryptophan hydrochloride using the procedure described above for the synthesis of LLV.
7

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
The methyl esters were purified by flash chromatography using hexanes/ethyl acetate (3:2), and the final
1
products were purified by flash chromatography using hexanes/ethyl acetate/acetic acid (7:11:0.2). H
NMR (CDCl3, ppm) δ: 8.33 (1 H, s), 7.55 (1 H, d, J
=
8.0 Hz), 7.32 (1 H, d, J
= 8.4 Hz), 7.17 (1 H, t, J =
7.0 Hz), 7.10 (1 H, t, J 7.6 Hz), 6.97 (1 H, s), 6.10 (1 H, d, J
=
=
8.0 Hz), 5.35 (4 H, m), 4.92 (1 H, q, J =
6.0 Hz), 3.33 (2 H, m), 2.77 (2 H, t, J
= 6.3 Hz), 2.05 (6 H, m), 1.49 (2 H, m), 1.40-1.14 (14 H, m), 0.88 (3
H, t, J
=
6.8 Hz). 13C NMR (CDCl3, ppm) δ: 175.3, 174.2, 136.1, 130.3, 130.1, 128.1, 127.9, 127.8, 123.2,
122.2, 119.7, 118.4, 111.4, 109.6, 53.4, 36.4, 31.5, 29.6, 29.3, 29.2, 29.1, 27.2, 27.1, 25.6, 25.4, 22.6,
14.1. HRMS (ESI, orbitrap) m/z: [M-H]– Calcd for C29H41O3,N2 465.3123; Found 465.3128.
2.5. Surface Tension Measurements
Surface tension was determined using an Attension Sigma 703D tensiometer (Bolin Scientific)
using a Wilhelmy plate. Solutions were prepared in 50 mM borate, pH 9.0, at least 30 min prior to
making measurements. After immersion of the plate in the each test solution, 10 min were allowed to
elapse in order for the reading to stabilize.
2.6. Kinetics
Formation of the conjugated diene in the products was monitored spectrophotometrically at 234 nm
(234 = 2.5 × 104 M-1 cm-1) at 25oC using either a Beckman DU 640 spectrophotometer (Table 1 and Figure
2) or a Hitachi U2900 spectrophotometer (Table 2). Substrate solutions were prepared in 50 mM sodium
borate, pH 9.0, and reactions were initiated by addition of enzyme. The initial rates were determined
from the first 10% of the reaction and fit to the Michaelis-Menten equation by nonlinear regression using
Sigma Plot.
2.7. Preparative Reaction of LLV with SBLO-1
A 100-mL solution of 132 M LLV and 3.2 nM SBLO-1 in 50 mM borate, pH 9.0, was stirred
vigorously at room temperature until the A234 stopped increasing (ca. 15 min). The reaction mixture was
acidified to pH 1 with concentrated phosphoric acid and extracted with ether (3 × 20 mL). Concentration
of the extracts yielded an oil, which was dissolved in 5.0 mL of ethanol and treated with 11 mg of NaBH4
8

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
at 0oC. The mixture was stirred for 30 min at 0oC and 30 min at room temperature, and acetic acid was
added cautiously until gas evolution ceased. Water (12 mL) was added, and the solution was extracted
with ether (3 × 10 mL). Concentration of the ether extracts yielded an oil, which was dissolved in
methanol and treated with ethereal diazomethane (generated from 1-methyl-3-nitro-1-nitrosoguanidine
using an Aldrich micro diazomethane generator). Evaporation of the solvents produced an oil. HPLC of
this material gave the chromatogram shown in Figure 4 (traces A and B). Approximately 1 mg of this
substance was incubated for 1 h at room temperature with 10 L of pyridine and 10 L of BSTFA to
convert the hydroxyl group to its trimethylsilyl derivative. After evaporation of the pyridine and BSTFA
under a stream of nitrogen, the residue was dissolved in 1.0 mL of methanol, and the solution was stirred
under H2 for 1 h with 10 mg of 5% Pd on CaCO3. The catalyst was removed by centrifugation, and the
supernatant was analyzed by GC/MS to obtain the data in Figure 3.
2.8. Chromatography
Normal-phase HPLC was carried out using a 250 × 4.6 mm Alltech Adsorbosphere 5column
eluted isocratically at 1.0 mL/min using the mobile phases indicated in the Results section. UV detection
of diene hydroperoxides was carried out at 234 nm. Chiral-phase HPLC was performed on a 250 × 4.6
mm Daicel Chiralpak AD-H column. GC/MS analyses were carried out on a Hewlett/Packard GCD
instrument with a 12m × 0.3 mm HPI capillary column. The column temperature was maintained at 50oC
for 3 min and then increased from 50–250oC at 20o/min.
2.9. Stereochemical Analysis
The configurations of the carbon-13 oxygenated products from LLV and LDV were determined by
the procedure previously described for the products from linoleyldimethylamine [34].
2.10. Hydrolysis of products from LLT and LDT
Oxygenations of LLT and LDT by SBLO-1 and subsequent reduction of the products with NaBH4
were carried out by the same procedure described above for LLV. Approximately 0.5 umole (estimated
spectrophotometrically) of unpurified product dissolved in ethanol was added to a reaction vial, and the
9

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
ethanol was evaporated under a stream of nitrogen. The residue was dissolved in 1.0 mL of 3.0 M NaOH,
and the vial was sealed and heated for 14 h at 120oC. After cooling, the reaction mixture was acidified to
pH 3 with phosphoric acid and extracted with two 1-mL portions of dichloromethane. The
dichloromethane extracts were concentrated to dryness and dissolved in hexanes for analysis by normal-
phase HPLC.
3. Results
3.1. Surface Tension Measurements
Surface-tension measurements were carried out to determine whether the N-linoleoylamino acids in
Scheme 2 undergo significant aggregation at the concentrations to be used in the kinetics studies.
Figure 1 presents surface tension measurements for solutions of N-linoleoyl-D-valine (0.75–96 M) and
N-linoleoyl-D-trptophan (0.38–48 M) in 50 mM borate buffer, pH 9.0. Surface tension is plotted against
the log of the concentration. The plot for N-linoleoyl-D-valine gives a straight line, which implies that
significant aggregation does not occur over this concentration range. Similar results were obtained with
N-linoleoyl-L-valine (Figure S2). In contrast, the plot for N-linoleoyl-D-tryptyophan (Figure 1) can be
fit to two intersecting lines, and the point
of intersection corresponds to a
concentration of 6.4 M, which is taken as
the critical micelle concentration (CMC)
[41]. Surface tension measurements with
N-linoleoyl-L-tryptophan gave a similar
plot (Figure S3) with the point of
intersection at 7.7 M. These results
indicate that the CMC of the N-
linoleoyltryptophan enantiomers is about
6–8 M.
10

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
3.2. Kinetics
Table 1 presents kinetics results for N-linoleoyl-L-valine (LLV), N-linoleoyl-D-valine (LDV) and
linoleic acid as substrates for SBLO-1 in 50 mM borate, pH 9.0, at 25oC. Both LLV and LDV are very
good substrates, with values of kcat/Km that are slightly higher than the value for linoleic acid. The kinetic
parameters for LLV and LDV are nearly equal, and the values of kcat/Km are identical within error.
Table 2 presents the initial rates of oxygenation of N-linoleoyl-D-tryptophan (LDT) and N-
linoleoyl-L-tryptophan (LLT) at a substrate concentration of 2.0 M, which is below the critical micelle
concentration. Under these conditions, the D enantiomer is a better substrate than the L enantiomer by a
factor of 2.7. LDT is also a better substrate at higher concentrations (Figure 2). When the data are fit to
the Michaelis-Menten equation, LDT has apparent kcat of 165 ± 8 s-1 and an apparent Km of 5.1 ± 1.1 M;
the corresponding parameters for LLT are 122 ± 7 s-1 and 23 ± 3 M. The data indicate that aggregated
forms of LLT and LDT can increase the concentration of available substrate.
kcat (s–1)
kcat
(µM–1, s–1)
Substrate
Km (µM)
∕Km
rate
LDT
LLT
0.82 ± 0.04
0.31 ± 0.03
linoleic acid
LLV
220 ± 13
205 ± 6
182 ± 7
13.3 ± 1.4
8.9 ± 0.7
7.8 ± 0.7
16.5 ± 2.1
23 ± 2
LDV
24 ± 3
Table 2. Initial rates of oxygenation of
2.0 µM LLT and LDT by 2.0 nM SBLO-1
at 25°C in 50 mM sodium borate, pH 9.0
Table 1. Kinetic parameters for the oxygenation of substrates by
SBLO-1 in 50 mM sodium borate, pH 9.0, 25°C.
11

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
3.3. Product Analysis
Incubation of SBLO-1 with LLV gave rise to an absorption at 234 nm characteristic of a conjugated
diene, and the intensity of the absorption indicated about 95% conversion to diene product (assuming 234
= 2.5 x 104 M-1cm-1, the value for 13-HPOD). Following treatment with NaBH4 (to reduce the
hydroperoxides to alcohols) and methylation with diazomethane, the NMR spectrum of the product shows
the resonances expected for either methyl 13-hydroxy-9(Z),11(E)-octadecadienylvaline or methyl 9-
hydroxy-10(E),11(Z)-octadecadienylvaline. To distinguish between these regioisomers, the crude product
was methylated with diazomethane, catalytically hydrogenated, trimethylsilylated, and analyzed by
GC/MS. The major product (95%) gave a mass spectrum (Figure 3A) consistent with the presence of a
trimethylsiloxy group on carbon-13. A minor product (5%) gave a mass spectrum consistent with the
presence of a trimethylsilyloxy group on carbon-9 (Figure 3B). These results indicate that the action of
SBLO-1 on LLV produces primarily 13-hydroperoxide.
The methyl esters of the NaBH4-reduced product from LLV were also analyzed by normal-phase
HPLC (hexanes/isopropanol, 95:5) with uv detection at 234 nm (Figure 4, traces A and B).
12

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
In addition to the major product (I), which eluted at 11 min, minor products (II–IV) were detected at 14
min, 19 min and 22 min. The relative intensities of these peaks are shown in Table 3. Four peaks with the
same retention times were obtained on a synthetic mixture obtained by nonenzymatic autoxidation of the
methyl ester of LLV followed by treatment with NaBH4 (Figure 4, trace C). The four peaks in the
synthetic mixture had about equal intensity. NMR spectra of the isolated components of the synthetic
mixture indicated that the materials eluting at 11 min and 19 min were E,Z isomers and the other two peaks
appear to be E,E. The presence of small amounts of E,E isomers in the product mixture from the
oxygenation of linoleic acid by SBLO-1 was previously reported by Gardner [19], and these products have
also been detected in our laboratory.
Peak I from the enzymatic reaction was collected and analyzed by chiral-phase HPLC
(heptane/ethanol, 80:20, 0.8 mL/min). A major product (98%) was observed at 27 min and a minor
product (2%) at 21 min (Figure 5A). A chromatogram of
methyl 13-hydroxy-9(Z),11(E)-octadecadienoyl-L-valine
from the nonenzymatic autoxidation showed the same two
peaks (Figure 5B), but in this case, the intensities were
equal, as expected for a racemic mixture. These results
indicate that the enzymatic reaction gives primarily one
stereoisomer. The configuration at carbon-13 for the
I
II
III
IV
LLV 92.9 ± 0.3 1.5 ± 0.1 3.1 ± 0.9 2.5 ± 0.6
LDV 95.0 ± 0.9 1.1 ± 0.5 2.7 ± 0.4 1.3 ± 0.1
Table 3. Relative intensities of the HPLC peaks from the
analysis of products from oxygenation of linoleoyl-L-valine
(LLV) and linoleoyl-D-valine (LDV). I–IV refer to the labeled
peaks in Figure 4. Values are the average of two experiments
(± average deviation). The samples of LLV and LDV used in
these experiments were freshly purified by flash chromato-
graphy and shown by UV spectrometry to contain < 1% diene
hydroperoxide. No products were detected in a control experi-
ment from which SBLO-1 was omitted.
13

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
major enantiomer was determined to be S using a previously published method [34], as described in the
Supplementary Content.
Using the methods described above, the major product obtained from LDV was shown to be 13(S)-
hydroperoxy-9(Z),11(E)-octadecadienoyl-D-valine. HPLC analysis of the methylated, NaBH4-treated
product mixture gave a product distribution that was similar to that obtained from LLV (Table 3), and
chiral-phase HPLC of the major product indicated that the 13(S):13(R) ratio was 97:3. These results
indicate that the stereochemical configuration of the valine moiety does not appreciably affect the
products produced by SBLO-1.
Oxygenation of LLT by SBLO-1 followed by reduction with NaBH4 and methylation gave one
major product (retention time = 13.9 min) by normal-phase HPLC (hexanes/isopropanol (93:7), 1.0
mL/min). The 1H-NMR spectrum of the HPLC-purified methyl ester was consistent with either the 13-
hydroxy or 9-hydroxy products. The trimethylsilyl derivative of the methyl ester was not sufficiently
volatile for GC/MS analysis. To establish the regiochemistry of the product, the amide bond was cleaved
by alkaline hydrolysis. Analysis of the hydrolysis products by normal-phase HPLC
(hexanes/isopropanol/acetic acid, 96:4:0.1) revealed a peak with the same retention time (5.9 min) as 13-
hydroxy-9(Z),11(E)-octadecadienoic acid (13-HODE) and only a very small peak (< 5% of the major
peak) at the retention time (7.7 min) of 9-hydroxy-10(E),12(Z)-octadecadienoic acid (9-HODE). The
hydrolysis products were esterified with diazomethane and subjected to chiral-phase HPLC
(hexanes/ethanol, 95:5). This gave peaks at the retention times of methyl 13(S)-HODE (9.4 min) and
methyl 13(R)-HODE (7.6 min) in a ratio of 93:7. These results establish that the major product from
oxygenation of LLT by SBLO-1 is the C-13 hydroperoxide and has the S configuration. Analogous
experiments with LDT indicated that the oxygenation occurred primarily (95%) at carbon-13 with a
13(S):13(R) ratio of 94:6.
4. Discussion
SBLO-1 shows little or no stereoselectivity towards the two enantiomers of N-linoleoylvaline. The
values of kcat/Km for LLV and LDV are identical within error, and the distributions of regioisomeric and
14

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
stereoisomeric products are not appreciably different. For both enantiomers, the major product is the
13(S) hydroperoxide, as in the case with linoleic acid. This result is consistent with the hypothesis that
the linoleoyl moieties of LLV and LDV bind in the same manner as linoleic acid.
The kcat/Km values for the N-linoleoylvaline derivatives are about 40% higher than the value for
linoleic acid, owing to a reduction in Km. These results suggest that the valine moiety makes a small
favorable contribution to the affinity for the enzyme. The fact that addition of the valine moiety increases
rather than decreases kcat/Km is most readily accommodated by the hypothesis the valine moiety binds
largely external to the protein. The modest size of the increase and the lack of stereoselectively suggest
that the interaction of the valine group with the protein is weak.
Reactions catalyzed by SBLO-1 are thought to proceed through a pentadienyl radical bound at the
active site (Scheme 1). Differences in the manner in which this intermediate is bound might influence its
reactions with O2 in a manner that affects the distribution of regioisomers or stereoisomers that are
formed. The similar product ratios for the LLV and LDV indicate that the stereochemical configuration
of the valine moiety has little effect on the binding of the intermediates. With both LLV and LDV, minor
products are formed with the same retention times as the E,E isomers. The appearance of these isomers
suggests that the pentadienyl radical might dissociate from the enzyme on a small fraction of turnovers
and react reversibly with O2 in solution [42]. If dissociation of the intermediate occurs, it does so to about
the same extent with both enantiomers, since the percentages of these minor products are not appreciably
different.
In order to explore the effects of an amino acid moiety with a larger side chain, we investigated the
L and D enantiomers of N-linoleoyltryptophan. These studies were complicated by the finding that these
substances have a relatively low critical micelle concentration. Below the CMC, LDT is a better substrate
than LLT by a factor of 2.7. For a reaction proceeding through a transition state, a rate difference of this
magnitude would indicate that the enzyme binds the transition state for the oxygenation of LDT more
favorably than the transition state for oxygenation of LLT by 0.6 kcal/mol. Since the reaction catalyzed
15

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
by SBLO-1 involves tunneling, this interpretation is not strictly applicable, but it serves to indicate that
the effect of the stereochemical configuration of the tryptophan moiety is small. Interpretation of this
difference by modeling is likely to be challenging, since as discussed below, the most likely binding site
for the tryptophan moiety is near helix-2, which has been shown to have considerable conformational
mobility [33].
Taken together, the results imply that the interaction of SBLO-1 with the amino acid moieties in the
substrates we have studied is weak and not highly sensitive to stereochemistry. The results are consistent
with the hypothesis that the amino acid moiety binds externally to the enzyme with limited interaction
with the surface of the protein. This interpretation also accommodates our finding that the rates of
oxygenation of LLT and LDT continue to rise above the CMC. Aggregated forms of these substances
could associate with the surface of the enzyme and allow the linoleoyl side chain to enter the active site.
Interestingly, the small degree of stereoselectivity in favor of monomeric LDT is still observed in the
aggregated forms.
Gaffney and coworkers have studied the interaction of SBLO-1 with substrate analogues that
contain a spin label at the polar end [32]. EPR studies of the complexes formed by these spin labels with
the enzyme indicate that the polar ends bind at the surface of the protein in a region at the interface of
helix-2 and helix-11. The polar groups bearing the spin labels appear to have a high degree of mobility
[32,43]. This finding suggests that the polar groups in these substrate analogues interact only weakly
with the surface. The results in this study are consistent with the hypothesis that N-linoleoylamino acid
substrates bind in a similar manner.
Acknowledgements
We are grateful to Erik Ralph for contributing to the development of some of the methods used in
this study. This work was supported by a grant from the National Science Foundation (RUI CHE-
1213262) and by the Stephen G. Hobar Memorial Research Fund, the Drs. Anthony and Joyce D. Kales
Undergraduate Research Fund, and the Bucknell Program for Undergraduate Research.
16

N-Linoleoylamino Acids as Chiral Probes
of Substrate Binding by Soybean Lipoxygenase-1
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