5 S,15 S-Dihydroperoxyeicosatetraenoic Acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1

Article abstract of DOI:10.1021/acs.biochem.8b00889

The reaction of 5S,15S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA₄ when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB₄. For 12-LOX and 5,15-diHpETE, the kinetic parameters are k_cat = 0.17 s^-1 and k_cat/K_M = 0.011 μM^-1 s^-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are k_cat = 4.6 s^-1 and k_cat/K_M = 0.21 μM^-1 s^-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB₄ from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC₅₀ of 1.3 μM; however, LXB₄ did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB₄ is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA₄ is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA₄ and LXB₄ are produced by 12-LOX but only if LTA₄ is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

Full text of DOI:10.1021/acs.biochem.8b00889

Subscriber access provided by UNIV OF LOUISIANA
Article
5S,15S-Dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) as a
Lipoxin Intermediate: Reactivity and Kinetics with Human Leukocyte 5-
Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-Lipoxygenase-1
Abigail R. Green, Cody J. Freedman, Jennyfer Tena, Benjamin E.
Tourdot, Benjamin Liu, Michael Holinstat, and Theodore Russell Holman
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00889 • Publication Date (Web): 08 Nov 2018
Downloaded from http://pubs.acs.org on November 14, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 27
Biochemistry
1
1
2
3
4
5
5S,15S-Dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) as a Lipoxin Intermediate: Reactivity and
Kinetics with Human Leukocyte 5-Lipoxygenase, Platelet 12-Lipoxygenase, and Reticulocyte 15-
6
7
8
Lipoxygenase-1
Abigail R. Green1, Cody Freedman1, Jennyfer Tena1, Benjamin E. Tourdot2, Benjamin Liu1, Michael Holinstat2,
Theodore R. Holman1*
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1Department of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High Street, Santa Cruz CA
2
95064, USA Department of Pharmacology, University of Michigan, 500 South State Street, Ann Arbor, MI 48109,
USA
*Corresponding author: Tel: 831-459-5884. Email: holman@ucsc.edu
FUNDING: NIH R01 GM105671 (MH and TRH) and NIH R01 HL11405 (MH and TRH).
NIH F32 HL129491 (BET).
ACS Paragon Plus Environment

Biochemistry
Page 2 of 27
2
1
2
3
4
5
6
7
8
9
Abbreviations: LOX, lipoxygenase; 15-LOX-1, human reticulocyte 15-lipoxygenase-1; sLO-1, soybean lipoxygenase-
1; 5-LOX, leukocyte 5-lipoxygenase; 12-LOX, human platelet 12-lipoxygenase; GP, glutathione peroxidase; AA,
arachidonic acid; HETE, hydoxy-eicosatetraenoic acid; HpETE, hydroperoxy-eicosatetraenoic acid; diHETEs,
dihydroxy-eicosatetraenoic acids; 5-HETE, 5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 5-HpETE, 5-hydro
peroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid; 12-HpETE, 12-hydroperoxy-5Z,8Z,11E,14Z-eicosatetraenoic acid; 15-
HpETE, 15-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid; 5,15-HETE, 5S,15S-dihydroxy-6E,8Z,11Z,13E-
eicosatetraenoic acid; 5,15-diHpETE, 5,15-dihydroperoxy-6E,8Z,11Z,13E-eicosatetraenoic acid; 5,6-diHETE, 5S,6R-
dihydroxy-7E,9E,11Z,14Z-eicosatetraenoic acid; LTA4, 5S-trans-5,6-oxido-7E,9E,11Z,14Z-eicosatetraenoic acid;
LTB4, 5S,12R-dihydroxy-6Z,8E,10E,14Z-eicosatetraenoic acid; LipoxinA4 (LXA4), 5S,6R,15S-trihydroxy-7E,9E,
11Z,13E-eicosatetraenoic acid; LipoxinB4 (LXB4), 5S,14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 27
Biochemistry
3
1
2
3
Abstract
4
5
The reaction of 5,15-diHpETE with human 5-lipoxygenase (LOX), human platelet 12-LOX and
6
7
human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing
lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is
the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4.
For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/KM = 0.011 μM-1s-1 (106-
fold and 1600-fold lower than for 12-LOX oxygenation of AA, respectively). On the other hand, for 15-
LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/KM = 0.21 μM-1s-1 (3-fold higher and similar to
that for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of
reaction of 15-LOX-2 with 5,15-diHpETE (Biochemistry 55, 2832-2840, 2016). Our data indicate that 12-
LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet
aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM, however, LXB4 did not
significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary
product of 12-LOX and 15-LOX-1 catalysis if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold
more efficient than 12-LOX. LXA4 is the primary product with 5-LOX, but only if 15-HpETE is the
substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX, but only if LTA4
is the substrate, as described previously (Biochimica et Biophysica Acta 1133, 223-234, 1992).
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Biochemistry
Page 4 of 27
4
1
2
3
Introduction:
4
5
6
7
8
9
Lipoxins (LXs) were discovered in 19841, 2 and are a class of specialized pro-resolving mediators
(SPMs) derived from arachidonic acid (AA), which are involved in inflammation resolution. The first two
LXs discovered were lipoxinA4 (LXA4) and lipoxinB4 (LXB4), with the epi-lipoxins, 15-epi-LXA4 and 15-
epi-LXB4, being discovered later.3 The initial step of acute inflammation is to recruit macrophages with the
generation of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). Eventually, SPMs, such as
LXs, are generated via transcellular pathways to reverse the inflammatory response and initiate cellular
repair.4 Researchers have investigated the biosynthetic pathway for the production of LXs and the generally
accepted biosynthesis of LXs requires two distinct enzymatic reactions with AA. The first step is the
oxygenation of AA to form a hydroperoxide, which is then converted to an epoxide. This can be
accomplished by either 5-LOX or 15-LOX, generating either the 5,6-epoxide or the 14,15-epoxide,
respectively. The second step is oxygenation at the other end of the molecule from the epoxide, adding a
hydroperoxide moiety. There is then a subsequent hydrolysis of the epoxide and reduction of the
hydroperoxide, creating either the 5,6,15-trihydroxy- or the 5,14,15-trihydroxy-eicosatetraenoate, depending
on the initiating LOX isozyme (Figure 1).5, 6 The specific pathway depends on the cell type involved, either
with one cell type that has both LOXs, or a transcellular biosynthetic pathway, which involves the transfer of
an intermediate between two cell types. Another biosynthetic pathway for LXs involves 5-LOX in
neutrophils and 12-LOX in platelets. The hypothesis is that 5-LOX generates LTA4 in the neutrophil, which
is then transferred to the platelet, where 12-LOX subsequently abstracts a hydrogen atom from C13 of LTA4,
with oxygen attacking C15. The epoxide ring is subsequently opened by a hydrolase, generating a
delocalized cation, which is attacked by water, generating either LXA4 with a C6 attack or LXB4 with a C14
attack (Figure 1).7, 8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Another pathway to making LXs, involves 5,15-diHpETE as an intermediate. A hydrogen atom is
abstracted from C10 of 5,15-diHpETE, generating a delocalized radical, which can convert either the C5
hydroperoxide or the C15 hydroperoxide to an epoxide (5,6- or 14,15-epoxide). Subsequent hydrolysis of the
ACS Paragon Plus Environment

Page 5 of 27
Biochemistry
5
1
2
expoxide and reduction of the hydroperoxide generates either the 5,6,15-trihydroxy- or the 5,14,15-
trihydroxy-eicosatetraenoates. Like the cation intermediate discussed above, both LXA4 and LXB4 can be
generated in this manner since the resulting radical is delocalized over 9 carbons due to the full conjugation
of the tetraene system (Figure 1).9 The reactivity of this chemical step is enhanced by the fact that 5,15-
diHpETE is an “activated” intermediate. The energy required to abstract the C10 hydrogen atom from 5,15-
diHpETE is markedly lower than the hydrogen atom from AA or 5-hydroperoxyeicosatetraenoic acid (5-
HpETE), due to the greater conjugation of its intermediate, 9-carbons for 5,15-diHpETE versus 5-carbons for
AA.9 Therefore, the ease of hydrogen atom abstraction from 5,15-diHpETE could enhance the rate of either
LXA4 or LXB4 biosynthesis.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1: Biosynthetic pathways for LX production. The carbon whose hydrogen atom is abstracted is listed next to the
LOX isozyme, such as C7, C10 or C13. For the conversion of 5,15-DiHpETE to the two lipoxins, the LOX isozyme is not
specified because that is the subject of this publication.
This last biosynthetic pathway is supported by fact that the LX intermediate, 5,15-diHpETE, is
observed in a variety of biological samples. One of the earliest observations of 5,15-diHpETE was in
ACS Paragon Plus Environment

Biochemistry
Page 6 of 27
6
1
2
3
4
5
6
7
8
9
activated porcine leukocytes, when either AA or 15-HpETE were added as precursors.10 5,15-diHpETE was
also observed with isolated porcine 5-LOX, which reacted with 15-HpETE, at a 30% rate of that with AA.11
5,15-diHpETE could then be further modified to LXB4 with purified rabbit reticulocyte 15-LOX-1 (r15-
LOX-1), presumably due to an epoxide intermediate.12 r15-LOX-1 could also convert 15-HETE to LXB4,
which was due to double oxygenation at C5 and C15, as evidenced by labeled O2 being found on both
carbons.12, 13 Porcine leukocyte 12-LOX also converts 5,15-diHpETE to LXB4, which was deduced by the
observation of four isomers of LXB4, due to the non-enzymatic hydrolysis of 5-OH,14,15-LTA4.14 LXB4 was
also produced by incubation of 15-HpETE with human leukocytes, with labeled O2 incorporating into only
C5, and not C14, indicating the presence of a hydrolyzed 14,15-epoxide.15 5,15-diHpETE can also be
generated by the addition of AA to human eosinophils, where both 15-LOX-1 and 5-LOX are present.16
Previously, we have shown that human epithelial 15-LOX-2 does not react with 5,15-diHpETE and
thus it is not implicated in producing LXs though this intermediate.9 In the present study, we expand this
investigation into the biosynthetic pathway of LXs generation from 5,15-diHpETE by comparing the relative
activities of human 5-LOX, human platelet 12-LOX and human reticulocyte 15-LOX-1. These results will
help establish a better understanding of the relative roles of these three LOX isozyme in the biosynthesis of
LXs from 5,15-diHpETE.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Methods
Chemicals. The lipid mass spectrometry standards, 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid
(5,15-diHETE), 5S,6R,15S-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid (LipoxinA4 (LXA4)), and 5S,
14R,15S-trihydroxy-6E,8Z,10E,12E-eicosatetraenoic acid (LipoxinB4 (LXB4)), were purchased from
Cayman Chemical. Arachidonic acid (AA) was purchased from Nu Chek Prep, Inc. and used to synthesize
5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE), 13S-hydrperoxy-9Z,11E-octadecadienoic acid
(13-HPODE), 5S,15S-hydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5,15-diHETE), 5S-hydro-15S-peroxy-
6E,8Z,11Z,13E-eicosatetraenoic acid (5OH,15-HpETE), and 5S,15S-dihydroperoxy-6E,8Z,11Z,13E-
eicosatetraenoic acid (5,15-diHpETE). Synthesis of 5-HETE, 13HPODE, and 5,15-diHpETE were performed
ACS Paragon Plus Environment

Page 7 of 27
Biochemistry
7
1
2
3
as previously described with 5-LOX and AA, 15-LOX-1 and LA, 15-HPETE and 5-LOX, respectively.9, 17 18
,
4
5
6
7
8
9
The synthesis of 5,15-diHETE was performed as done for 5,15-diHpETE, with the exception that
trimethylphosphite was added in 2:1 molar excess as a reductant prior to the HPLC purification. 5OH,15-
HpETE was synthesized from 5-HETE as follows. 60μM of 5-HETE was reacted in 1 L of 100mM
NaBorate, pH 9.2 and 4 mg of lipoxygenase from soybean (Sigma). This reaction was monitored at 254 nm
to completion, quenched with 1% (v/v) glacial acetic acid, extracted with 1 L of DCM, and evaporated to
dryness. The 5OH,15-HpETE was then purified isocratically via high performance liquid chromatography
(HPLC) on a Higgins Haisil Semi-preparative (5μm, 250mm x 10mm) C18 column with 45:55 of 99.9%
acetonitrile, 0.1% acetic acid and 99.9% water, 0.1% acetic acid. Purity was assessed via liquid
chromatography-mass spectrometry to be greater than 90%.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Expression and Purification of 15-LOX-1, 12-LOX, and 5-LOX. Overexpression and purification of
wild-type human reticulocyte 15-lipoxygenase-1 (15-LOX-1), platelet 12-lipoxygenase (12-LOX), and 5-
lipoxygenase (5-LOX) were performed as previously described.17, 19, 20 The purity of 15-LOX-1 and 12-LOX
were assessed by SDS gel to be greater than 85%, and metal content was assessed on a Finnigan inductively-
coupled plasma-mass spectrometer (ICP-MS), via comparison with iron standard solution. Cobalt-EDTA
was used as an internal standard. 5-LOX was isolated as an impure ammonium sulfate precipitant from E.
coli expression due to its dramatic loss of activity upon isolation.
Product Analysis of LOX isozyme reactions with 5,15-diHpETE, 5-OH,15-HpETE, and 5,15-diHETE.
15-LOX-1 (120 nM), 12-LOX (250 nM), or 5-LOX (25mg of ammonium sulfate precipitated protein) was
reacted in 12 mL of 25 mM HEPES, pH 7.5, at ambient temperature, with 10 μM of oxylipin (5,15-
diHpETE, 5OH,15-HpETE, or 5,15-diHETE) for one hour and quenched with 0.5% glacial acetic acid and 5
μM of 7-hydroxy-docosahexaenoic acid as an extraction standard. 3 μM of 13-HPODE was added to the
reactions with 5,15-diHETE to activate iron as described in.21 Each quenched reactions was extracted with
12 mL of DCM, resuspended in 1 mL methanol and split 2:1, with the 30% portion being reduced with
trimethylphosphite, while the other 60% was left unreduced. The samples were then evaporated under a
ACS Paragon Plus Environment

Biochemistry
Page 8 of 27
8
1
2
3
4
5
6
7
8
9
stream of N2 to dryness, reconstituted in 50 μL of methanol, and further diluted with 50 μL of 0.1% formic
acid in water prior to UPLC-MS/MS analysis. Control reactions without enzymes were also conducted and
used for background subtraction, ensuring oxylipin degradation products were removed from analysis.
Chromatographic separation was performed on a Dionex UltiMate 3000 UHPLC with a C18 column
(Phenomenex Kinetex, 1.7 m, 150mm x 2.1mm). The autosampler was held at 4o C and injection volume
was 90 uL. Mobile phase A consisted of water with 0.1% (v/v) formic acid and mobile phase B was
acetonitrile with 0.1% formic acid. Flow rate was 0.400 mL/min. The initial condition (30% B) was
maintained for 2.3 minutes. Mobile phase B was then ramped to 65% over 28.7 minutes, held at 65% for 1
minute, ramped to 100% over 0.1 min, held at 100% for 7 minutes, and finally returned to 30% to equilibrate
for 7 minutes. The chromatography system was coupled to a Dionex Ultimate-3000 DAD and Velos Pro
linear ion trap (Thermo Scientific) for UV and mass spectrum analysis. Analytes were ionized via heated
electrospray ionization with -4.0 kV spray voltage, 60, 10, and 0 arbitrary units for sheath, auxiliary and
sweep gas, respectively. The RF amplitude of the S-Lens was 49%, and the probe and capillary temperatures
were 50o C and 380o C, respectively. All analyses were performed in negative ionization mode at Normal
resolution setting. MS2 was performed at 35% normalized collision energy in a targeted manner with a mass
list containing the following m/z ratios + 0.1: 319.2, 335.2, 343.2, 351.2, 367.2, 383.2, and 399.2. Products
were identified by matching retention times, UV spectra and fragmentation patterns to known standards. Or
in the cases where MS standards were not available, structures were deduced from comparison to known and
theoretical fragments.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Steady State Kinetics of 5-LOX, 12-LOX and 15-LOX-1 with 5,15-diHpETE, 12-LOX with 5,15-
diHETE. 15-LOX-1 and 5-LOX reactions were performed, at ambient temperature, in a 1 cm2 quartz cuvette
containing 2 mL of 25 mM HEPES, pH 7.5 with substrate (AA, 15HpETE, or 5,15-diHpETE). 12-LOX
reactions were also performed in a 1 cm2 quartz cuvette containing 2 mL of 25 mM HEPES with substrate
(AA, 5,15-diHpETE, or 5,15-diHETE), and the pH was 8.0. Additionally, 12-LOX reactions with 5,15-
diHETE contained 3 μM 13-HPODE and 5-LOX reactions contained 200 M ATP. AA concentrations were
ACS Paragon Plus Environment

Page 9 of 27
Biochemistry
9
1
2
varied from 0-29 μM, 15-HpETE concentrations were varied from 0-80 M, 5,15-diHpETE concentrations
were varied from 0-45 μM, and 5,15-diHETE concentrations were varied from 0-130 μM. 5-LOX reactions
were compared using 10 M substrate for AA, 15-HpETE, and 5,15-diHpETE with product formation per
time analyzed on UV-Vis as well as LC/MS. Concentration of AA was determined by measuring the amount
of 15-HpETE produced from complete reaction with soybean lipoxygenase-1 (sLO-1). Concentrations of
5,15-diHpETE and 5,15-diHETE were determined by absorbance at 247 nm. Reactions were initiated by the
addition 15-LOX-1 (120 nM) or 12-LOX (250 nM) or 5-LOX (approx. 200 nM of crude AS preparation) and
were monitored on a Perkin-Elmer Lambda 45 UV/VIS spectrophotometer. Product formation was
determined by the change in absorbance at 234 nm for 15-HpETE/5HpETE (ε234nm = 25,000 M-1 cm-1), 5,15-
diHpETE (ε254nm = 21,90000 M-1 cm-1),9 and 302 nm for lipoxins (ε234nm = 50,000 M-1 cm-1). KaleidaGraph
(Synergy) was used to fit initial rates (at less than 20% turnover), as well as the second order derivatives
(kcat/KM) to the Michaelis-Menten equation for the calculation of kinetic parameters.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5,15-diHpETE and LXB4 titration into human platelets. The University of Michigan Institutional Review
Board approved all research involving human volunteers. Washed human platelets (250ꢀµL at 3.0 x108
platelets/mL) were dispensed into glass cuvettes and incubated with 0-10 µM of either LXB4 or 5,15-
diHpETE for 10 minutes at 37°C. Oxylipin-treated platelets were then stimulated with 0.25 µg/mL of
collagen (Chrono-log), stirring at 1100 rpm and 37°C, in a Chrono-log Model 700D lumi-aggregometer and
platelet aggregation was recorded for six minutes.
In order to determine the biosynthetic products of 5,15-diHpETE ex vivo, platelets were isolated
from human whole blood via serial centrifugation and adjusted to 3.0 x 108 platelets/mL in Tyrode’s buffer
(10 mM HEPES, 12 mM NaHCO3, 127 mM NaCl, 5 mM KCl, 0.5 mM NaH2PO4, 1 mM MgCl2, and 5 mM
glucose), as previously published.22 One ml of platelets was incubated with varying concentrations of 5,15-
diHpETE (0-20 µM) or vehicle (DMSO) for 10 minutes at 37 C, and then the platelets were pelleted by
centrifugation at 1000 x g for 1 minute. The supernatant was transferred to a fresh tube and snap frozen.
Oxylipins were extracted and analyzed via UPLC-MS/MS as described previously,9 with the following
ACS Paragon Plus Environment

Biochemistry
Page 10 of 27
10
1
2
3
4
5
exceptions. The m/z transitions for 5,15-diHETE, LTB4-d4, RvD2-d5, and LipoxinB4 were 335.2115,
339.2197, 380.5141, and 351.2221, respectively.
6
7
8
Results and Discussion
9
Lipoxin synthesis from 5,15-diHpETE, 5-OH,15-HpETE, and 5,15-diHETE. To study the conversion of
5,15-diHpETE to LX through product identification, 5,15-diHpETE was reacted with human 5-LOX, 12-
LOX, and 15-LOX-1. For the reaction of 5-LOX with 5,15-diHpETE, no activity was detected at 302 nm
(the absorbance of conjugated lipoxins) and LXA4 and LXB4 were both undetected via LC-MS. This data
indicates that 5,15-diHpETE is a poor substrate for 5-LOX in vitro. In order to determine the relative
turnover of 5,15-diHpETE with 5-LOX, the rate of AA, 15-HpETE and 5,15-diHpETE were compared under
the same reaction conditions. 5-LOX converted AA to 5-HpETE at 5 ng/sec and 15-HpETE to 5,15-
diHpETE at 0.5 ng/sec, but there was no conversion of 5,15-diHpETE to LXA4 observed (the detection limit
of the experiment is 0.0003 ng/sec). This data indicates that the oxidation reaction of 5-LOX with 5,15-
diHpETE is more than 1700-fold slower than that with 15-HpETE. This was unexpected because 5-LOX
abstracts the C10 hydrogen atom from 5-HpETE to generate leukotriene A4, (LTA4) which is the same
hydrogen atom presented by 5,15-diHpETE. In addition, the hydrogen atom on C10 for 5,15-diHpETE is
more reactive than that of 15-HpETE, due to conjugation,9 making this lack of reactivity even more
noteworthy. However, 5-LOX is able to convert 15-HpETE into LXA4 at a rate of 0.5 ng/sec. This difference
in reactivity of 5,15-diHpETE versus 15-HpETE is most likely due to a difference in the rate of substrate
capture, as defined by Northrop et al.23, 24 Previously, we have shown that the kcat for generating LTA4 is the
same when starting with arachidonic acid (AA) or 5-HpETE. However, the kcat/KM is 10-fold slower for 5-
HpETE than AA, due to a slow binding step.17 We propose that 5-LOX’s lack of reactivity with 5,15-
diHpETE is due to an even slower binding step for 5,15-diHpETE relative to 5-HpETE, limiting reactivity
completely. We therefore propose that 5-LOX produces LXA4 from 15-HpETE by generating 5,15-diHpETE
in situ and subsequently generates LXA4 without releasing the 5,15-diHpETE intermediate, similar to the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mechanism for LTA4 synthesis.
ACS Paragon Plus Environment

Page 11 of 27
Biochemistry
11
In contrast, both 12-LOX and 15-LOX-1 reacted with 5,15-diHpETE, manifesting observable rates at
1
2
3
4
302 nm, which is consistent with LXs being formed. LC-MS analysis of the reduced products confirmed that
both 12-LOX and 15-LOX-1 made LXB4 (RT = 8.8 min), but not LXA4 (Figure 2, dashed traces). We also
observe LXB4 isoforms, with the all-trans-LXB4 (RT = 8.1 min), 14S-LXB4-epimer (RT = 8.9 min), and the
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2: UPLC-UV-Vis chromatogram of h12-LOX (A) and h15-LOX-1 (B) products from 5,15-diHpETE. The wavelength
of 302 nm is the absorbance maximum for lipoxins (Supplemental S1). At 302 nm, 11 peaks are observed in the unreduced
(solid line) reaction that coalesce to 2 clusters of peaks upon reduction (dashed line). Based on the UV-Vis spectrum, retention
times, parent masses, and MS/MS spectra (Supplemental S2), the identities of these peaks have been determined to be all-
trans-LxB4 and its 14S-epimer at 7.9 min, LxB4 and its 14S-epimer at 8.6 min and 5,14,15-trihydroperoxy-LxB4 at 13.6 min. The
peaks at 9.5, 9.8, 10.0, 10.3 and 11.5 min are the monohydroperoxide products and the peaks at 11.3, 12.2, and 13.2 min are
the dihydroperoxide products. *Note: although only one double bond stereochemistry is depicted for these compounds, the
differing retention times suggest rearrangements. MS/MS spectra contain fragments that indicate the hydroperoxide moiety’s
position may differ between these mono- and dihydroperoxy-HETEs.
14S-all-trans-LXB4-epimer (RT = 8.2 min). These products are synthesized by the opening of the epoxide
via dehydration, as previously observed with purified rabbit 15-LOX-1 and 15-HpETE as the substrate.12
ACS Paragon Plus Environment

Biochemistry
Page 12 of 27
12
1
2
3
4
5
6
7
8
9
However, some of these products could also be formed by the competing reaction, oxygenation. In order to
differentiate these two reaction mechanisms, the reaction products were analyzed without reductant added
during product extraction (Figure 2, solid traces). Both dehydration (i.e. epoxide formation) and oxygenation
require abstraction at C10, with migration of the radical to C14. Dehydration occurs upon reaction of the
radical with the hydroperoxide in an anti-periplanar orientation, while oxygenation occurs with antarafacial
attack of the radical by molecular oxygen. Both reactions require the active site ferrous-water species to
reduce the radical intermediate and form the final product. Comparing reduced and un-reduced products can
contribute to differentiating the two sets of products, hydroperoxides and alcohols, produced by oxygenation
and dehydration, respectively. Analysis of the data clearly demonstrates that hydroperoxides are present, due
to the fact that many of the peaks migrated to shorter retention times upon reduction. Specifically, the tri-
hydroperoxy species (5,14,15-triHpETE, RT = 13.6 min, Figure 2) is observed, which could only be formed
via oxygenation at C14. It should be noted that while we observe the parent mass of 5,14,15-triHpETE (399
m/z, hydrogen loss in the negative mode), we also observe the neutral loss of water from 5,14,15-triHpETE
(Supplemental S2), resulting in the most intense ion for 5,14,15-triHpETE at 381 m/z. Additionally, because
there are three hydroperoxide moieties, multiple peaks that correspond to the loss of up to three waters upon
ionization (Supplemental S1 & S2) are observed. The additional hydroperoxide peaks correspond to both
oxygenation products, which have lost an oxygen, most likely due to reduction during the long reaction time
in buffer. These products include: 5-OOH,14-OOH,15-OOH-triHpETE (RT = 13.7 min), 5-OOH,14-OH,15-
OOH-triHETE, 5-OH,14-OOH,15-OOH-triHETE, 5-OH,14-OH,15-OOH-triHETE, 5-OOH,14-OOH,15-
OH-triHETE, and 5-OH,14-OOH,15-OH-triHETE. These products exhibit multiple peaks, which most likely
correspond not only to the hydroperoxide position in these molecules, but also to the all-trans, S-epimers,
and other isomers resulting from the non-enzymatic epoxide opening at C14 and C15. The non-enzymatic
epoxide opening is also supported by comparing the ratio of the sum of all-trans-LXB4, 14S-LXB4 and 14S-
all-trans-LXB4 to LXB4 in the reduced samples of 5,15-diHpETE (LxB4 isomers and epimers/LxB4 = 0.91 +
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.2) and 5,15-diHETE (LxB4 isomers and epimers = 0.19 + 0.02) as substrates with 12-LOX. The
ACS Paragon Plus Environment

Page 13 of 27
Biochemistry
13
1
2
dehydration and oxygenation peaks have been quantified, demonstrating no preference for dehydration over
oxygenation, with 12-LOX having a dehydration/oxygenation ratio of 1.3 + 0.2 and 15-LOX-1 having a ratio
of 1.2 + 0.2 (Figure 2, supplemental S2). Since only LXB4 is produced by 12-LOX and 15-LOX-1 from
5,15-diHpETE, with it being generated either through oxygenation or dehydration (i.e. via the epoxide), the
data indicates similar LX reactivities for both LOX isozymes. It should be noted that due to the reactive
nature of these di- and tri-hydroperoxides, the data was scanned for Hock rearrangement decomposition,
however, no evidence was observed for these products.25
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To eliminate the possibility of epoxide generation, 12-LOX and 15-LOX-1 were reacted with 5,15-
diHETE, the reduced form of 5,15-diHpETE, which is incapable of forming epoxides. 12-LOX reacted with
5,15-diHETE producing LxB4 and its isoforms, but 13-HPODE was required. 13-HPODE is a known
oxidant of lipoxygenase that reduces lag times and expands the substrate range of lipoxygenases by
activating the LOX isozyme when the endogenous hydroperoxide product cannot oxidize the intermediate
ferrous ion.18, 26 For this particular reaction, the conversion of 5,15-diHETE to LXB4 implies that 12-LOX
oxygenates at C14 because the 14,15-epoxide cannot be formed from the alcohol. The requirement of 13-
HpODE is because the ferrous-water intermediate cannot efficiently reduce the hydroperoxide radical at
C14, thus releasing the radical intermediate and generating the inactive ferrous-water species. The ferrous-
water intermediate is subsequently oxidized by 13-HpODE, generating the active form of 12-LOX. This
conclusion is supported by the comparison of the reduced and un-reduced products (Figure 3). The un-
reduced reaction manifests a large peak (RT = 9.8 min), which elutes after LxB4 (RT = 8.8 min). This peak
has an MS/MS fragmentation pattern of a C14 hydroperoxy moiety (supplemental S3), which becomes LxB4
upon reduction, as seen by its change in retention time and its MS/MS fragmentation (supplemental S3) and
confirms an oxygenation reaction on C14. It should be noted that we observe a large reduction in the number
of isoforms of LxB4, as compared to the reaction with 5,15-diHpETE as the substrate. This is appropriate
given that this is a region- and stereo-specific oxygenation reaction and no epoxide was formed that could
undergo non-enzymatic hydrolysis to generate the LxB4 isoforms.
ACS Paragon Plus Environment

Biochemistry
Page 14 of 27
14
1
2
3
4
5
6
7
8
9
Interestingly, 15-LOX-1 cannot perform an oxygenation reaction with 5,15-diHETE, even with 13-
HPODE present, indicating that 15-LOX-1 cannot oxygenate at C14 with 5,15-diHETE as the substrate. This
is unexpected since 15-LOX-1 can oxygenate 5,15-diHpETE. Therefore, the hydroxides at C5 and C15
appear to change substrate binding so that either the hydrogen atom abstraction at C10 is prohibited or the
oxidation of the active site ferrous-water intermediate to the active ferric-hydroxide moiety by the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 3: UPLC-UV-Vis chromatogram of h12-LOX products from 5,15-diHETE. The wavelength of 302 nm is the
absorbance maximum for lipoxins (Supplemental S1). At this wavelength, three peaks, at times 8.1, 8.8, and 9.8 min, are
observed in the unreduced reaction (solid line) that coalesce to two peaks, 8.1 and 8.8 min, upon reduction (dashed line).
Based on the UV-Vis spectrum, retention times, parent mass, and MS/MS spectra (Supplemental S2), the identities of these
peaks have been determined to be all-trans-LxB4 at 8.1, LxB4 at 8.8 and 14-hydroperoxy-LxB4 at 9.8 min.
hydroperoxyl radical is prohibited. This hypothesis is supported by the fact that hydroperoxyl moieties in
these eicosanoids behave more hydrophobically than hydroxyl moieties, as evident by their longer retention
on a C18 column (Supplemental S1). In addition, 15-LOX-1 will not oxygenate 15-HETE either, but it will
oxygenate 15-HpETE, indicating a difference in reactivity with the reduced oxylipin.
ACS Paragon Plus Environment

Page 15 of 27
Biochemistry
15
It should be reiterated that 15-LOX-1 and 12-LOX do not make LXA4 from 5,15-diHpETE, however,
1
2
3
4
they do make LXB4. This is an interesting observation considering that the radical intermediate of 5,15-
diHpETE can potentially form either the 5,6- or 14,15-epoxide. However, the active sites of both 12-LOX
and 15-LOX-1 preferentially generate only the 14,15-epoxide. This restricted reactivity could be related to
the fact that the oxygen tunnel for both 15-LOX-1 and 12-LOX is located near C14, as observed by the C14
oxygenation products.27, 28 Therefore, the opening to the oxygen tunnel in the active site could provide a dual
function. Primarily to oxygenate 5,15-diHpETE but also to provide the space for the 14,15-epoxide to form,
thus giving the oxygen tunnel an added functionality.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Biochemistry
Page 16 of 27
16
1
2
3
4
5
6
7
8
9
Steady-state kinetics of 15-LOX-1 and 12-LOX with 5,15-diHpETE and 12-LOX with 5,15-
diHETE. Considering that hydrogen atom abstraction is typically rate-limiting in lipoxygenase reactions,29-31
the steady-state kinetics of 15-LOX-1 and 12-LOX with 5,15-diHpETE revealed an interesting difference
between their relative rates. When reacted with AA, 12-LOX abstracts the hydrogen atom at C10, generating
more than 95% 12-HpETE.19 On the other hand, 15-LOX-1 abstracts the hydrogen atom at C10 only ≈10%
of the time, with C13 being the major target.32-34 We confirmed this result by observing approximately 10%
of 12-HpETE and 90% of 15-HpETE being generated from AA reacted with 15-LOX-1. Therefore, 12-LOX
has a 78-fold larger kcat/KM than 15-LOX-1 for abstracting the hydrogen atom from C10 of AA and
generating 12-HpETE, 18 μM-1s-1 and 0.23 μM-1s-1, respectively (Table 1). It should be noted that these 15-
LOX-1 kinetic values for C10 abstraction are estimated from the total rate observed for 15-LOX-1 and its
product percentages. This indicates that 12-LOX is more efficient in both product release (kcat) and substrate
capture (kcat/KM) than 15-LOX-1 in abstracting a hydrogen atom from C10 on AA (13-fold for kcat and 78-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 1: Steady State Parameters for 15-LOX-1 and 12-LOX reaction with 5,15-diHpETE and
5,15-diHETE
Enzyme
Substrate
kcat (s-1)
Km (M)
kcat/Km(M-1s-1)
12-LOX
15-LOX-1
AA (C10 abstraction)
AA (C10 abstraction )
18 + 0.8
1.4 + 0.1*
1.0 + 0.1
6 + 1
18 + 2
0.23 + 0.03*
12-LOX
15-LOX-1
5,15-diHpETE
5,15-diHpETE
0.17 + 0.007
4.6 + 0.1
14 + 1
23 + 2
0.011 + 0.007
0.21 + 0.01
12-LOX
15-LOX-1
5,15-diHETE**
5,15-diHETE**
0.18 + 0.02
nr***
113 + 19
nr
0.0015 + 0.0001
nr
* kcat and kcat/Km values were confirmed daily relative to the kcat and kcat/Km values of AA. The C10
abstraction kinetic values of 15-LOX-1 are 10% of the AA kinetic values, which include both the 15-
HpETE and 12-HpETE kinetics (kcat and kcat/Km for AA are 14 + 1s-1 and 2.3 + 0.3M-1 s-1, respectively)
** 3M of 13-HPODE is added to activate 12-LOX.
*** nr = no reaction
fold for kcat/KM). However, despite the preference of 12-LOX for abstracting at C10 on AA, 15-LOX-1 has
both a better product release rate (kcat) and substrate capture rate (kcat/KM) than 12-LOX, for abstracting the
ACS Paragon Plus Environment

Page 17 of 27
Biochemistry
17
1
2
C10 hydrogen atom from 5,15-diHpETE (Table 1). For kcat, the 12-LOX rate for C10 abstraction from 5,15-
diHpETE is 106-fold less than that of AA (kcat = 0.17 s-1), while the 15-LOX-1 rate is 3.3-fold greater than
that of C10 abstraction from AA (kcat = 4.6 s-1). For kcat/KM, the 12-LOX rate for C10 abstraction from 5,15-
diHpETE is 1600-fold less than that of AA (kcat/KM = 0.011 μM-1s-1), while the kcat/KM for 15-LOX-1 is the
same as that of C10 abstraction from AA (kcat/KM = 0.21 μM-1s-1). Effectively, there is no loss of activity for
15-LOX-1 abstracting the hydrogen atom from C10 of 5,15-diHpETE relative to AA, but 12-LOX has a
large loss of activity with 5,15-diHpETE relative to AA, as seen by a significant decrease in kcat and kcat/KM
(Table 1). This data indicates that the hydroperoxy moieties on C5 and C15 of 5,15-diHpETE, disrupt its
positioning in the 12-LOX active site for C10 abstraction significantly, while not affecting 15-LOX-1
appreciably. It should be noted that for both 12-LOX and 15-LOX-1, only 10% of the total 5,15-diHpETE in
the reaction is converted to LXB4 and addition of 12-LOX, 15-LOX-1 or 5,15-diHpETE at 10% conversion
did not improve the turnover percentage. This implies that there is some form of suicide product inhibition,
which is a common observation in LOX reactions, due to the reactive nature of the radical intermediates, and
has previously been observed with 12-LOX and LTA4.35, 36
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Biochemistry
Page 18 of 27
18
1
2
3
4
5
6
7
8
9
As noted above, LX formation from the reduced intermediate, 5,15-diHETE, is only observed with
12-LOX in the presence of 13-HPODE, and no rate was observed for 15-LOX-1. However, the rate of 12-
LOX with 5,15-diHETE is extremely slow, manifesting a kcat/KM that is 10-fold less than that seen for 5,15-
diHpETE (kcat/KM = 0.0015 μM-1s-1). Since the kcat of 12-LOX with 5,15-diHETE is essentially the same as
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4: 5,15-diHpETE, but not LXB4, inhibits collagen-mediated platelet aggregation. Isolated human platelets were treated
with increasing concentrations of either LXB4 or 5,15-diHpETE for ten minutes and then stimulated with collagen (0.25 µg/mL). A)
Representative tracings of 5,15-diHpETE or LXB4 treated platelets stimulated with collagen. B) Data represents mean ± S.E.M.
Statistical analysis was performed comparing oxylipin concentration to vehicle control using one-way ANOVA with Dunnett’s
multiple comparison test. *P<0.05, **P<0.01, ***P<0.001.
its kcat for 5,15-diHpETE, this lower rate of substrate capture (kcat/KM) is manifested in a 10-fold increase in
KM, (113 + 19 μM). As discussed above, 12-LOX only oxygenates 5,15-diHETE but it oxygenates and
dehydrates 5,15-diHpETE. Since the kcat for both 5,15-diHETE and 5,15-diHpETE are similar, it indicates
that the rate of product release for both oxygenation and dehydration are also similar. This implies that the
reaction steps after the irreversible hydrogen abstraction step, such as the reduction of the radical
intermediate for oxygenation or dehydration by the ferrous-water moiety, are either not rate-limiting or
comparable in rate for the two reactions. It is also observed that the rate of substrate capture (kcat/KM)23 for
ACS Paragon Plus Environment

Page 19 of 27
Biochemistry
19
1
2
3
4
5
6
7
8
9
12-LOX is decreased by a factor of 10 for 5,15-HETE relative to 5,15-HpETE, indicating that there is a rate
difference in a reaction step prior to the irreversible hydrogen atom abstraction step, such as substrate
binding. As mentioned above, this could be due to its smaller size or the more polar hydroxyl moiety relative
to the hydroperoxide, as seen by its RP-HPLC retention time.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5,15-diHpETE and LXB4 titration into human platelets. To determine whether or not 5,15-diHpETE
inhibits platelet activation, platelets from 11 separate donors were treated with varying concentrations of
5,15-diHpETE (0.5-20 μM) or vehicle control (DMSO) prior to stimulation with collagen, a GPVI and α2β1
agonist. Compared to vehicle control, 5,15-diHpETE inhibited platelet aggregation in response to collagen
(0.25 µg/mL) stimulation with an IC50 of 1.3 μM (Figure 4). Furthermore, over 90% reduction in collagen-
Figure 5: Platelets produce LxB4 from 5,15-diHPETE. The amount of LxB4 produced by platelets treated with 5, 10 or 20
M of 5,15-diHPETE are plotted in this histogram. The standard deviation of each value is represented by error bars.
mediated platelet aggregation was observed in platelets treated with a concentration greater than 10 μM of
5,15-diHpETE. The antiplatelet effects of LXB4 were also investigated; however, in contrast to 5,15-
diHpETE, LXB4 did not significantly inhibit collagen-mediated platelet activation at the concentrations
tested.
With respect to the biosynthetic pathway, human platelets were treated with 5,15-diHpETE and
shown to produce LXB4 (Figure 5), but they did not produce LXA4. This is consistent with the in vitro
results, where purified 12-LOX only makes LXB4. However, the amount of LXB4 that we observe from 5,15-
diHpETE treated platelets is very small compared to the unreacted 5,15-diHpETE (less than 1.2 %),
indicating that this is not an efficient pathway for the generation of LXB4. In addition, since LXB4 does not
ACS Paragon Plus Environment

Biochemistry
Page 20 of 27
20
1
2
3
4
5
have a strong antiplatelet effect, it is reasonable to conclude that 5,15-diHpETE, and not its downstream
product, LXA4, is the active oxylipin species that affects platelet biology in our experiments.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 21 of 27
Biochemistry
21
In summary, the reaction of 5,15-diHpETE with various human lipoxygenase isozymes was
1
2
3
4
investigated to determine the relative rates of producing LXs. Previously, it was established that 15-LOX-2
did not react with 5,15-diHpETE, consistent with its enhanced substrate selectivity relative to the other LOX
isozymes.9 15-LOX-2 abstracts a hydrogen atom from C13 of AA, but does not abstract from C10, the only
available methylene carbon of 5,15-diHpETE. In the current work, it was determined that 5-LOX does not
react with 5,15-diHpETE either. However, 5-LOX can produce LXA4 if 15-HpETE is the substrate (Figure
6), indicating that substrate binding is impaired when the hydroperoxide is on both C5 and C15, consistent
with our previous work with 5-LOX where oxygenation of the oxylipin affects substrate binding
significantly.17 In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE (Figure 6), albeit at slow
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Biochemistry
Page 22 of 27
22
1
2
3
4
5
6
7
8
9
rates relative to AA. Surprisingly, 15-LOX-1 has a 27-fold greater kcat and a 20-fold greater kcat/KM than 12-
LOX. This result is unusual because C10 is the preferred location for hydrogen abstraction for 12-LOX, so it
appears that the active site of 12-LOX is less tolerant of oxygenated fatty acids than 15-LOX-1, which
lowers its catalytic rates, as already seen for 5-LOX.17 The low in vitro catalytic rate for 12-LOX with 5,15-
diHpETE is corroborated by the fact that when 5,15-diHpETE is added to platelets, LXB4 production is
extremely low (1.2 + 0.2 % total conversion), consistent with the poor in vitro LXB4 production rate. This
result is in contrast to the conventional wisdom that assumes 12-LOX is the biosynthetic enzyme for LXs
production from 5,15-diHpETE, due to its preference for abstracting a hydrogen atom from C10.8
Nevertheless, LXs are highly active molecules so low production levels in platelets could still have large
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 23 of 27
Biochemistry
23
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6: Reaction scheme for LXA4 and LXB4 production. The production of LXA4 and LXB4 is dependent on both the order
of LOX reactivity and the nature of the substrate. The carbon atom from which the hydrogen atom is abstracted is indicated below
the LOX isozyme label, such as C7, C10 or C13. The central pathway which involves 5(S),15(S)-diHpETE is the subject of this
publication.
effects on the cellular response, resulting in 12-LOX still being a potentially biosynthetically relevant LOX
isozyme for making LXs. For comparison, Serhan and coworkers determined that human 12-LOX reacted
with LTA4, producing both LXA4 and LXB4, with a Vmax of 24.5 nmol/min/mg (or a kcat of 0.031 sec-1) and a
Vmax/Km of 3.1 nmol/min/mg/uM (or a kcat/KM of 0.004 sec-1uM-1).35 These values are markedly slower than
the current observed rates of 15-LOX-1 with 5,15-diHpETE (150-fold slower for kcat and 50-fold slower for
kcat/KM), suggesting that 15-LOX-1 may be a more predominant source of LXs than 12-LOX, depending on
the cells involved. Another interesting difference between 5,15-diHpETE and LTA4 as LOX substrates is
that with LTA4, both LXA4 and LXB4 are made, but with 5,15-diHpETE as the substrate, only LXB4 is made.
ACS Paragon Plus Environment

Biochemistry
Page 24 of 27
24
1
2
3
4
5
6
7
8
9
Clearly, the mechanisms of LX production are distinct, which could help in the future identification of the
source of LX biosynthesis. For example, if 5,15-diHpETE is the substrate, LXB4 is the primary product of
12-LOX and 15-LOX-1 catalysis, whereas LXA4 is the primary product with 5-LOX. However, if LTA4 is
the substrate, then 12-LOX generates both LXA4 and LXB4. Therefore, it appears that cells have multiple
pathways for making LXs, depending on the available LOX isozyme and the available substrate, 5,15-
diHpETE or LTA4. We are currently investigating the molecular mechanism of these reactions with active
site mutants in order to gain better insights into the differences between these distinct LX biosynthetic
pathways.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supplemental information. The supplemental figures that are referenced in this article (S1, S2, S3 and S4)
along with their corresponding legends are available online. These materials are supplied free of charge at
http://pubs.acs.org.
ACS Paragon Plus Environment

Page 25 of 27
Biochemistry
25
1
2
3
REFERENCES
4
5
6
7
8
9
[1] Serhan, C. N., Hamberg, M., and Samuelsson, B. (1984) Trihydroxytetraenes: a novel series of compounds formed
from arachidonic acid in human leukocytes, Biochem Biophys Res Commun 118, 943-949.
[2] Serhan, C. N., Hamberg, M., and Samuelsson, B. (1984) Lipoxins: novel series of biologically active compounds
formed from arachidonic acid in human leukocytes, Proc Natl Acad Sci U S A 81, 5335-5339.
[3] Claria, J., and Serhan, C. N. (1995) Aspirin triggers previously undescribed bioactive eicosanoids by human
endothelial cell-leukocyte interactions, Proc Natl Acad Sci U S A 92, 9475-9479.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[4] Serhan, C. N., Chiang, N., and Van Dyke, T. E. (2008) Resolving inflammation: dual anti-inflammatory and pro-
resolution lipid mediators, Nat Rev Immunol 8, 349-361.
[5] Romano, M., Cianci, E., Simiele, F., and Recchiuti, A. (2015) Lipoxins and aspirin-triggered lipoxins in resolution of
inflammation, Eur J Pharmacol 760, 49-63.
[6] Back, M., Powell, W. S., Dahlen, S. E., Drazen, J. M., Evans, J. F., Serhan, C. N., Shimizu, T., Yokomizo, T., and
Rovati, G. E. (2014) Update on leukotriene, lipoxin and oxoeicosanoid receptors: IUPHAR Review 7, Br J
Pharmacol 171, 3551-3574.
[7] Edenius, C., Haeggstrom, J., and Lindgren, J. A. (1988) Transcellular conversion of endogenous arachidonic acid to
lipoxins in mixed human platelet-granulocyte suspensions, Biochem Biophys Res Commun 157, 801-807.
[8] Sheppard, K. A., Greenberg, S. M., Funk, C. D., Romano, M., and Serhan, C. N. (1992) Lipoxin generation by human
megakaryocyte-induced 12-lipoxygenase, Biochimica et Biophysica Acta 1133, 223-234.
[9] Green, A. R., Barbour, S., Horn, T., Carlos, J., Raskatov, J. A., and Holman, T. R. (2016) Strict Regiospecificity of
Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential, Biochemistry 55, 2832-
2840.
[10] Borgeat, P., Picard, S., Drapeau, J., and Vallerand, P. (1982) Metabolism of arachidonic acid in leukocytes:
isolation of a 5,15-dihydroxy-eicosatetraenoic acid, Lipids 17, 676-681.
[11] Ueda, N., Kaneko, S., Yoshimoto, T., and Yamamoto, S. (1986) Purification of arachidonate 5-lipoxygenase from
porcine leukocytes and its reactivity with hydroperoxyeicosatetraenoic acids, J Biol Chem 261, 7982-7988.
[12] Kuhn, H., Wiesner, R., Alder, L., Fitzsimmons, B. J., Rokach, J., and Brash, A. R. (1987) Formation of lipoxin B by
the pure reticulocyte lipoxygenase via sequential oxygenation of the substrate, Eur J Biochem 169, 593-601.
[13] Kuhn, H., Wiesner, R., Lankin, V. Z., Nekrasov, A., Alder, L., and Schewe, T. (1987) Analysis of the stereochemistry
of lipoxygenase-derived hydroxypolyenoic fatty acids by means of chiral phase high-pressure liquid
chromatography, Anal Biochem 160, 24-34.
[14] Ueda, N., Yokoyama, C., Yamamoto, S., Fitzsimmons, B. J., Rokach, J., Oates, J. A., and Brash, A. R. (1987) Lipoxin
synthesis by arachidonate 12-lipoxygenase purified from porcine leukocytes, Biochem Biophys Res Commun
149, 1063-1069.
[15] Serhan, C. N., Hamberg, M., Samuelsson, B., Morris, J., and Wishka, D. G. (1986) On the stereochemistry and
biosynthesis of lipoxin B, Proc Natl Acad Sci U S A 83, 1983-1987.
[16] Morita, E., Schroder, J. M., and Christophers, E. (1990) Identification of a novel and highly potent eosinophil
chemotactic lipid in human eosinophils treated with arachidonic acid, J Immunol 144, 1893-1900.
[17] Smyrniotis, C. J., Barbour, S. R., Xia, Z., Hixon, M. S., and Holman, T. R. (2014) ATP Allosterically Activates the
Human 5-Lipoxygenase Molecular Mechanism of Arachidonic Acid and 5(S)-Hydroperoxy-
6(E),8(Z),11(Z),14(Z)-eicosatetraenoic Acid, Biochemistry.
[18] Wecksler, A. T., Kenyon, V., Garcia, N. K., Deschamps, J. D., van der Donk, W. A., and Holman, T. R. (2009) Kinetic
and structural investigations of the allosteric site in human epithelial 15-lipoxygenase-2, Biochemistry 48,
8721-8730.
[19] Ikei, K. N., Yeung, J., Apopa, P. L., Ceja, J., Vesci, J., Holman, T. R., and Holinstat, M. (2012) Investigations of
human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation, J Lipid Res.
[20] Joshi, N., Hoobler, E. K., Perry, S., Diaz, G., Fox, B., and Holman, T. R. (2013) Kinetic and structural investigations
into the allosteric and pH effect on the substrate specificity of human epithelial 15-lipoxygenase-2,
Biochemistry 52, 8026-8035.
[21] Wecksler, A. T., Kenyon, V., Deschamps, J. D., and Holman, T. R. (2008) Substrate specificity changes for human
reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation, Biochemistry 47, 7364-7375.
ACS Paragon Plus Environment

Biochemistry
Page 26 of 27
26
1
2
3
4
5
[22] Tourdot, B. E., and Holinstat, M. (2017) Targeting 12-Lipoxygenase as a Potential Novel Antiplatelet Therapy,
Trends Pharmacol Sci 38, 1006-1015.
[23] Northrop, D. B. (1998) On the meaning of Km and V/K in enzyme kinetics, J. Chem. Ed. 75, 1153-1157.
[24] Northrop, D. B. (1999) Rethinking fundamentals of enzyme action, Adv Enzymol Relat Areas Mol Biol 73, 25-55,
ix.
[25] Gardner, H. W., and Plattner, R. D. (1984) Linoleate hydroperoxides are cleaved heterolytically into aldehydes by
a Lewis acid in aprotic solvent, Lipids 19, 294-299.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[26] Kuhn, H., Wiesner, R., Stender, H., Schewe, T., Lankin, V. Z., Nekrasov, A., and Rapoport, S. M. (1986)
Requirement of monohydroperoxy fatty acids for the oxygenation of 15LS-HETE by reticulocyte lipoxygenase,
FEBS Lett 203, 247-252.
[27] Knapp, M. J., Seebeck, F. P., and Klinman, J. P. (2001) Steric control of oxygenation regiochemistry in soybean
lipoxygenase-1, J Am Chem Soc 123, 2931-2932.
[28] Knapp, M. J., and Klinman, J. P. (2003) Kinetic Studies of Oxygen Reactivity in Soybean Lipoxygenase-1,
Biochemistry 42, 11466-11475.
[29] Glickman, M. H., and Klinman, J. P. (1995) Nature of Rate-Limiting Steps in the Soybean Lipoxygenase-1 Reaction,
Biochemistry 34, 14077-14092.
[30] Glickman, M. H., Wiseman, J. S., and Klinman, J. P. (1994) Extremely Large Isotope Effects in the Soybean
Lipoxygense-Linoleic Acid Reaction, J. Am. Chem. Soc. 116, 793-794.
[31] Segraves, E. N., and Holman, T. R. (2003) Kinetic investigations of the rate-limiting step in human 12- and 15-
lipoxygenase, Biochemistry 42, 5236-5243.
[32] Bryant, R. W., Bailey, J. M., Schewe, T., and Rapoport, S. M. (1982) Positional specificity of a reticulocyte
lipoxygenase. Conversion of arachidonic acid to 15-S-hydroperoxy-eicosatetraenoic acid, J Biol Chem 257,
6050-6055.
[33] Kuhn, H., Barnett, J., Grunberger, D., Baecker, P., Chow, J., Nguyen, B., Bursztynpettegrew, H., Chan, H., and
Sigal, E. (1993) Overexpression, Purification and Characterization of Human Recombinant 15-Lipoxygenase,
Biochim. Biophys. Acta 1169, 80-89.
[34] Kuhn, H. (2000) Structural basis for the positional specificity of lipoxygenases, Prostaglandins Other Lipid Mediat
62, 255-270.
[35] Romano, M., Chen, X. S., Takahashi, Y., Yamamoto, S., Funk, C. D., and Serhan, C. N. (1993) Lipoxin synthase
activity of human platelet 12-lipoxygenase, Biochem J 296 ( Pt 1), 127-133.
[36] Kishimoto, K., Nakamura, M., Suzuki, H., Yoshimoto, T., Yamamoto, S., Takao, T., Shimonishi, Y., and Tanabe, T.
(1996) Suicide Inactivation of Porcine Leukocyte 12-Lipoxygenase Associated with Its Incorporation of 15-
Hydroperoxy-5,8,11,13-Eicosatetraenoic Acid Derivative, Biochimica et Biophysica Acta - Lipids & Lipid
Metabolism 1300, 56-62.
ACS Paragon Plus Environment

Page 27 of 27
Biochemistry
27
1
2
3
TOC
4
5
For Table of Contents Use Only
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment