Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase

Biochemical and Biophysical Research Communications 519 (2019) 81e85

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Gly188Arg substitution eliminates substrate inhibition in arachidonate 11R-lipoxygenase ~ ldemaa, Maarja Lipp, Ivar Ja €rving, Nigulas Samel, Priit Eek* Kaspar Po Department of Chemistry and Biotechnology, Tallinn University of Technology, Akadeemia tee 15, 12618, Tallinn, Estonia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 16 August 2019 Accepted 23 August 2019 Available online 30 August 2019

Lipoxygenases (LOXs) are dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxyl derivates. These products are precursors for different lipid mediators which are associated with pathogenesis of various diseases such as asthma, atherosclerosis and cancer. Several LOXs suffer from substrate inhibition, a potential regulatory mechanism, yet it is unclear what is the cause of this phenomenon. One such enzyme is the coral 11R-LOX which displays a significant decrease in turnover rate at arachidonic acid concentrations above 30 mM. In this report, site-directed mutagenesis and inhibition assays were employed to shed light on the mechanism of substrate inhibition in 11R-LOX. We found that introduction of a positive charge to the active site entrance with Gly188Arg substitution completely eliminates the slow-down at higher substrate concentrations. Inhibition of 11R-LOX by its catalysis product, 11(R)-hydroperoxyeicosatetraenoic acid, suggests an uncompetitive mechanism. We reason that substrate inhibition in 11R-LOX is due to additional fatty acid binding by the enzyme:substrate complex at an allosteric site situated in the very vicinity of the active site entrance. © 2019 Elsevier Inc. All rights reserved.

Keywords: Lipoxygenase Allosteric regulation Uncompetitive inhibition Eicosanoid Inflammation

1. Introduction Lipoxygenases (LOXs)1 are a class of non-heme iron containing dioxygenases that stereo- and regiospecifically peroxidize polyunsaturated fatty acids [1e3]. Mammalian LOXs catalyze the hydroperoxidation of arachidonic acid (AA, 20:4u6) into hydroperoxyl derivates which subsequently can be converted into lipid mediators such as leukotrienes, lipoxins and hepoxilins [4,5]. They have been implicated in various cellular processes, for instance in proliferation, differentiation and homeostasis, ultimately leading to serious pathologies including asthma, atherosclerosis and cancer. Animal LOXs consist of two distinctive domains: a smaller, Ca2þand membrane-binding regulatory N-terminal PLAT domain, and a larger, mainly a-helical C-terminal catalytic domain [1,6]. The catalysis by LOX enzymes involves a non-heme iron that cycles between ferric (Fe3þ) and ferrous (Fe2þ) forms to allow the formation of a fatty acid carbradical intermediate, which is subsequently peroxidized through sterically controlled addition of molecular oxygen

* Corresponding author. E-mail address: [email protected] (P. Eek). 1 LOX, lipoxygenase; AA, arachidonic acid; LA, linoleic acid; ETYA, eicosatetraynoic acid; HpETE, hydroperoxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid. https://doi.org/10.1016/j.bbrc.2019.08.132 0006-291X/© 2019 Elsevier Inc. All rights reserved.

[6,7]. The active site is shielded inside the catalytic domain and is situated in the center of a curved hydrophobic cavity e the substratebinding channel. The characteristics of this channel such as its shape, depth and polarity determine the positioning and orientation of the fatty acid substrate in relation to the active site iron, and therefore dictate the catalytic specificity of each LOX isoform [1,8,9]. The entrance to the substrate-binding channel has proven to be an important regulatory feature in LOXs. The putative access route is situated between helix a2 and a well-conserved arched helix, which forms the ceiling of the active site cavity (Fig. 1). Helix a2 and the adjoining protein chain are much less conserved and can be found in multiple conformations in the available LOX crystal structures. In the model of Plexaura homomalla 8R-LOX:AA complex, for instance, a2 is a long 7-turn helix that leaves the substrate channel orifice open [10]. In contrast, the a2 of the stabilized form of human ALOX5 makes only three turns and is shifted towards the active site, blocking the entrance entirely with a Phe177/Tyr181 “cork” [11]. The arachidonate 11R-LOX from the soft coral Gersemia fruticosa shares about 33% of its sequence with human ALOX5 e the key enzyme in the production of potent pro-inflammatory mediators, leukotrienes [12]. We determined the crystal structure of 11R-LOX, which revealed its a2 region in a strikingly similar arrangement to ALOX5, with Phe185 completely blocking the access to the active site [13]. Since 11R-LOX requires both Ca2þ and a lipid bilayer for

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20:4u6) were from Cayman Chemicals, phospholipids were from Avanti Polar Lipids, soybean LOX-1 was from Serva. All other chemicals were from Sigma. 2.2. Protein production

Fig. 1. The crystal structure model of 11R-LOX with the regulatory PLAT domain in green and the catalytic domain in light blue [13]. The active site is enclosed beneath the conserved arched helix (orange). The entrance to the substrate-binding channel is blocked by Phe185, which precedes the short a2 helix (dark blue). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

any in vitro activity [14] and the protein was crystallized in the absence of these components, we reasoned that the enzyme is inactive in solution due to being in a closed state. Only once 11RLOX is bound to a lipid membrane in a Ca2þ-induced manner it establishes an open, productive conformation. This process likely involves a cascade of conformational changes that start in the PLAT domain and are relayed to the a2 region [15], and might also be affected by the dimerization of the enzyme [16]. Several LOXs suffer from severe substrate inhibition including human ALOX5 [17,18], ALOX12 [19] and ALOX15B [20], but also coral 11R-LOX [15]. The catalytic activity of the coral enzyme decreases rapidly at AA concentrations higher than 30 mM. Rabbit ALOX15 does not normally display this phenomenon, but the removal of a positively charged moiety from the active site entrance (R403L substitution) gave rise to pronounced substrate inhibition [21]. A similar observation was made on coral 8R-LOX in which R182A mutation triggered the same behavior [10]. It should be noted that Arg182 of 8R-LOX is situated on helix a2, just next to the active site entrance. In 11R-LOX, neither of the aforementioned positions carry a positively charged residue as illustrated by the sequence alignment (Fig. 2). Instead, there is Gly188 that begins the short a2 helix, and a hydrophobic Leu415 (Fig. 1). In this study, we probed the key positions at the active site entrance using site-directed mutagenesis to understand their regulatory role in 11R-LOX. We also analyzed the kinetic properties of the enzyme in the presence of non-substrate fatty acids. The results provide new insight into the possible mechanism of substrate inhibition in LOXs.

Recombinant G. fruticosa 11R-LOX with an N-terminal His4 tag in pET-11a vector was produced as reported in Refs. [13,15]. In brief, mutations were introduced using whole-plasmid PCR with primers containing desired mismatches. Enzyme variants were expressed in 0.5-l cultures of Escherichia coli using the autoinduction method [22]. The protein was purified from the cell lysate with Ni-affinity chromatography, followed by anion exchange and size-exclusion steps. Purified enzymes were stored at 80  C. 2.3. Kinetic studies Activity assays of 11R-LOX variants were performed as in Refs. [13,15]. Briefly, catalysis was monitored at 236 nm on a UV1601 spectrophotometer (Shimadzu) at 20  C. Reaction mixture contained 50 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2, pH 8.0 buffer, small unilamellar vesicles (total phospholipid content ~60 mM), and AA in the range of 2e150 mM. Enzyme concentration was 6e27 nM depending on the specific activity of the enzyme variant. Reaction velocity was determined by the linear portion of the ascending part of the absorption curve. The kinetic parameters kcat and Km were calculated using the non-linear regression method in Prism v5.0 (Graphpad) software. In case of substrate inhibition, only the ascending part of the kinetic curve was used for calculations. In inhibition assays, 11(R)-hydroperoxyeicosatetraenoic acid (11RHpETE), 13(S)-hydroxyoctadecadienoic acid (13S-HODE) or ETYA was added to the mixture prior to initiation with the enzyme. The substrate and inhibitors were dissolved in ethanol. The amount of ethanol added to the reaction mixture was kept constant (1% v/v). 2.4. 13S-HODE production Mixture of 0.9 mM LA and 260 nM soybean LOX-1 in 50 mM Tris-HCl, pH 9.0 buffer was stirred for 7 min at room temperature. Hydroperoxyl groups were reduced to hydroxyls with the addition of SnCl2 to final concentration of 10 mM. After 5 min, the mixture was acidified to pH 3e4 with KH2PO4eHCl (1:1 molar ratio) and products were extracted with ethyl acetate. 13S-HODE was purified using thin layer chromatography on a silica gel plate eluted with hexane/diethyl ether/acidic acid (4:3:0.05), and extracted from the plate with methanol. 2.5. 11R-HpETE production

2. Materials and methods 2.1. Chemicals AA, linoleic acid (LA, 18:2u6) and eicosatetraynoic acid (ETYA,

Reaction was carried out in 50 mM Tris-HCl, 100 mM NaCl, 2 mM CaCl2, pH 8.0 buffer with ~60 mM phospholipids as small unilamellar vesicles and 13 nM 11R-LOX at room temperature. AA was added 4 times in 50-mM increments at 2-min intervals. After 9 min

Fig. 2. Sequence alignment of G. fruticosa 11R-LOX, P. homomalla 8R-LOX and rabbit ALOX15. Arg182 of 8R-LOX and Arg403 of ALOX15 (red boxes and arrows) are key residues to prevent substrate inhibition in those enzymes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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of total incubation time, the mixture was acidified to pH ~4 with KH2PO4eHCl (1:1 molar ratio) and products were extracted with cold ethyl acetate. 11R-HpETE was purified on a 250  4.6 mm Phenomenex Luna 5 mm silica column on ice with hexane/isopropanol (97:3) at 1.5 ml/min, and monitored at 235 nm. Purified fractions were pooled and stored in dry ethanol under nitrogen at 80  C.

3. Results 3.1. Introducing a positive charge to the active site entrance abolishes substrate inhibition Both coral 8R-LOX and rabbit ALOX15 have a positively charged residue at the entrance of the active site that participates in substrate coordination [10,21]. 11R-LOX, however, lacks a positive charge in these key positions (Fig. 2). To test whether this is the cause of substrate inhibition, we replaced the corresponding residues in 11R-LOX e Gly188 and Leu415 e with an Arginine. Singlemutation variants were expressed and purified, and their catalytic properties were assayed. The kinetic curve in Fig. 3 demonstrates that while the turnover rate of the wild-type enzyme is strongly suppressed at higher substrate concentrations (>40 mM), the G188R mutant retains its activity and follows Michaelis-Menten kinetics. The mutation does not alter the kcat and Km values notably (Table 1) which is also illustrated by the good match of the kinetic curves. L415R substitution conveys no such effect and the enzyme behaves similarly to the wild-type with pronounced substrate inhibition. 11R-LOX needs both Ca2þ and a lipid surface for catalytic activity [12,14]. Its crystal structure in the absence of these factors revealed that this is likely due to the putative entrance being in a closed conformation [13]. The orifice is blocked by Phe185, which is similar to the “FY cork” described in human ALOX5. We introduced a F185A mutation in hope to relieve this constraint, however the only effect we observed was an almost 2-fold reduction in turnover rate. F185A mutant does not display any activity in the absence of Ca2þ and/or lipid membranes (data not shown), so evidently larger structural reorganization is necessary to allow catalysis. Nevertheless, the reduction in catalytic activity is in agreement with the hypothesis postulated on ALOX5, that the active site cork is not only needed to obstruct access, but it also participates in substrate binding once the enzyme is in an open conformation [23].

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Table 1 Kinetic parameters of 11R-LOX and its active site entrance mutants (±SE).

WT G188R L415R F185A

kcat, s1

K m, m M

kcat/Km

377 ± 25 413 ± 10 434 ± 23 225 ± 21

15 ± 2 19 ± 1 15 ± 2 19 ± 4

25 ± 2 22 ± 1 29 ± 3 12 ± 1

3.2. Inhibition by non-substrate fatty acids suggests an allosteric binding site Substrate inhibition is a long-known property of LOXs, however, we wanted to check whether there is any feedback by the product as well, since non-substrate fatty acids (including catalysis products) can affect the activity of LOXs [24e28]. Hence, we assayed the effect of 11R-HpETE on 11R-LOX catalysis by adding it to the reaction mixture in the concentration range of 2e20 mM (Table 2). The difference between wild-type enzyme activities in Tables 1 and 2 are due to batch-to-batch variability. The same batch was used for all inhibitory assays. We have not observed the accumulation of di-HpETEs while lipoxygenating AA with 11R-LOX (data not shown), so utilization of 11R-HpETE as a substrate is negligible. In the case of a practically non-reversible reaction, one would expect that product accumulation inhibits an enzyme competitively, that is, by competing with the substrate for the active site. This should result in the increase of the apparent Km value. However, we observed that increasing the product concentration impacts mainly the apparent turnover rate, which is lowered (Table 2). The apparent Km also displays a slight trend, but instead of increasing it tends to decrease. Such an effect (reduction in both kcat and Km) can be attributed to uncompetitive inhibition. In a Lineweaver-Burk plot (Fig. 4) this is characterized by the lines that represent different inhibitor concentrations being parallel to each other, while in competitive and non-competitive modes the lines converge on either axis. Uncompetitive inhibition occurs if the inhibitor binds only the enzyme-substrate complex, locking the complex into an unproductive state until the inhibitor dissociates. The obtained data allows us to speculate that 11R-HpETE can act on 11R-LOX in an uncompetitive manner, in which case the enzyme must have an additional fatty acid binding site. 13S-HODE, which is the reduced product of 15-LOX catalysis using LA as a substrate, displays a similar effect, but the impact on the apparent kcat is even larger so a mixed inhibition mode could be considered. The irreversible LOX inhibitor ETYA has no effect on the apparent Km value, impacting only the turnover rate, which is consistent with its previously reported mode of action [29].

Table 2 Apparent kinetic parameters of wild-type 11R-LOX in the presence of fatty acid inhibitors (±SE).

Fig. 3. Kinetic curves of 11R-LOX and its active site entrance mutants. The curves are drawn in the data range that was used for fitting to the Michaelis-Menten equation. Error bars denote standard deviation (n ¼ 3).

AA þ 11R-HpETE 2 mM 5 mM 7.5 mM 10 mM 20 mM þ 13S-HODE 20 mM 40 mM þ ETYA 20 mM 40 mM 80 mM

kcat, s1

K m, m M

kcat/Km

283 ± 13

14 ± 1

20 ± 1

249 ± 8 245 ± 12 193 ± 9 157 ± 5 174 ± 6

12 ± 1 15 ± 2 10 ± 1 7.8 ± 0.6 12 ± 1

21 ± 1 16 ± 1 19 ± 1 20 ± 1 15 ± 1

139 ± 16 62 ± 6

11 ± 3 8.4 ± 2.0

12 ± 2 7.3 ± 1.1

168 ± 13 125 ± 5 119 ± 17

19 ± 3 15 ± 1 15 ± 4

9.0 ± 0.7 8.2 ± 0.4 7.9 ± 1.3

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Fig. 4. Lineweaver-Burk plots of 11R-LOX inhibition by a) its product, 11R-HpETE, b) the product and an allosteric effector of ALOX15, 13S-HODE, and c) the irreversible inhibitor ETYA. The plots were calculated using the kinetic parameters obtained by non-linear regression analysis. In the case of competitive or non-competitive inhibition, the lines converge on y- or x-axis, respectively (either apparent kcat or Km stays constant). Uncompetitive inhibition results in parallel lines (both apparent kcat and Km decrease).

4. Discussion There have been several explanations for substrate inhibition in LOXs, but the lack of definitive evidence has hindered the emergence of a uniform theory. In 11R-LOX, introduction of a positively charged moiety at the substrate channel entrance eliminates substrate inhibition. This correlates well with the results obtained with coral 8R-LOX, in which the corresponding reverse mutation, R182A,

induces severe inhibition [10]. The authors explained the data with a cooperative substrate inhibition model, and reasoned that the effect is due to an untethered substrate, which can assume several non-productive conformations in the active site. While their data fitted the cooperative model well, we find it difficult to understand how binding in a non-productive conformation can cause concentration-dependent inhibition assuming the substrate channel can accommodate only one fatty acid molecule at a time. Another possible mechanism for substrate inhibition is the dissociation of the intermediary fatty acid radical from the active site [30]. If the intermediate dissociates from the active site in the middle of the catalytic cycle, the non-heme iron remains in its ferrous form leaving the enzyme inactive until reactivated by another hydroperoxide. In such a state, substrate binding would be unproductive and block reactivation, and therefore, result in substrate inhibition. The fact that G188R substitution eliminates substrate inhibition in 11R-LOX supports this hypothesis. A positively charged Arginine at the active site entrance can provide an additional means to coordinate the substrate by its carboxylate head and prevent early dissociation since according to our mutational studies [13], this enzyme binds AA in an aliphatic tail-first fashion, and its carboxyl group remains at the entrance. Alternatively, there have been several reports of the allosteric nature of LOX enzymes. It has been suggested there is an allosteric inhibitory fatty acid binding site in soybean LOX-1 and human ALOX15 [24]. Catalysis products like 13S-HODE can evidently affect ALOX15 and also ALOX15B allosterically, since they impact the substrate selectivity of these enzymes [25,26]. Mass spectrometry data indicate that a single chain of human ALOX12 binds at least two molecules of AA [19]. Even though Arg403 at the substrate channel entrance of ALOX15 enzymes is believed to participate in substrate tethering [31,32], this is likely not the only role of the residue. While 13S-HODE induces transient dimerization of rabbit ALOX15 [33], no such effect was observed for the R403L mutant [21]. Dimerization of ALOX15 is thought to involve helix a2 [33]. Arg403, while not situated on that helix, might participate in inducing conformational changes in the a2 region that lead to dimerization and activation of the enzyme in a fatty acid-mediated manner [21]. This means that the dimerization-inducing allosteric fatty acid binding site of ALOX15 should be in the very vicinity of the active site entrance. Our data of using the catalysis product, 11R-HpETE, as an 11RLOX inhibitor suggest an uncompetitive inhibition mechanism, that is, the inhibitor binds only to the enzyme-substrate complex. We can assume that the same regulatory fatty acid binding site can be utilized by the substrate in which case the apparent outcome would be substrate inhibition. Since G188R mutation abolishes this effect in 11R-LOX, the inhibitory site should be close to the active site entrance like in ALOX15. Furthermore, the introduced charge might not only coordinate the bound substrate, but also obstruct additional fatty acid binding, which would otherwise result in a blocked enzyme-substrate complex. Our results together with previous findings demonstrate that the entrance to the substrate binding channel is a crucial regulatory part of LOX enzymes. Substrate inhibition, characteristic to several LOXs, is one of the features determined by the properties of this portal. Our data suggest that substrate inhibition in 11R-LOX can occur due to additional fatty acid binding at the active site entrance. The biological function of this phenomenon has been studied in several enzymes [34], but its role in LOXs is yet to be established.

Conflicts of interest The authors declare no conflicts of interest.

~ldemaa et al. / Biochemical and Biophysical Research Communications 519 (2019) 81e85 K. Po

Acknowledgements ~ugu for comments on the manuscript. This We thank Vello To work was supported by institutional research funding (IUT 19-9 to N.S.) of the Estonian Ministry of Education and Research. References [1] M.E. Newcomer, A.R. Brash, The structural basis for specificity in lipoxygenase catalysis, Protein Sci. Publ. Protein Soc. 24 (2015) 298e309, https://doi.org/ 10.1002/pro.2626. [2] H. Kühn, S. Banthiya, K. van Leyen, Mammalian lipoxygenases and their biological relevance, Biochim. Biophys. Acta 1851 (2015) 308e330, https:// doi.org/10.1016/j.bbalip.2014.10.002. [3] A.R. Brash, Lipoxygenases: occurrence, functions, catalysis, and acquisition of substrate, J. Biol. Chem. 274 (1999) 23679e23682. [4] R. Mashima, T. Okuyama, The role of lipoxygenases in pathophysiology; new insights and future perspectives, Redox Biol. 6 (2015) 297e310, https:// doi.org/10.1016/j.redox.2015.08.006. € m, C.D. Funk, Lipoxygenase and leukotriene Pathways : [5] J.Z. Haeggstro biochemistry , biology , and roles in disease, Chem. Rev. 111 (2011) 5866e5898. [6] I. Ivanov, D. Heydeck, K. Hofheinz, J. Roffeis, V.B. O'Donnell, H. Kühn, M. Walther, Molecular enzymology of lipoxygenases, Arch. Biochem. Biophys. 503 (2010) 161e174, https://doi.org/10.1016/j.abb.2010.08.016. [7] C. Schneider, D.A. Pratt, N.A. Porter, A.R. Brash, Control of oxygenation in lipoxygenase and cyclooxygenase catalysis, Chem. Biol. 14 (2007) 473e488, https://doi.org/10.1016/j.chembiol.2007.04.007. [8] R. Vogel, C. Jansen, J. Roffeis, P. Reddanna, P. Forsell, H.-E. Claesson, H. Kühn, M. Walther, Applicability of the triad concept for the positional specificity of mammalian lipoxygenases, J. Biol. Chem. 285 (2010) 5369e5376, https:// doi.org/10.1074/jbc.M109.057802. [9] M. Jisaka, R.B. Kim, W.E. Boeglin, A.R. Brash, Identification of amino acid determinants of the positional specificity of mouse 8S-lipoxygenase and human 15S-lipoxygenase-2, J. Biol. Chem. 275 (2000) 1287e1293. [10] D.B. Neau, G. Bender, W.E. Boeglin, S.G. Bartlett, A.R. Brash, M.E. Newcomer, Crystal structure of a lipoxygenase in complex with substrate: the arachidonic acid-binding site of 8R-lipoxygenase, J. Biol. Chem. 289 (2014) 31905e31913, https://doi.org/10.1074/jbc.M114.599662. [11] N.C. Gilbert, S.G. Bartlett, M.T. Waight, D.B. Neau, W.E. Boeglin, A.R. Brash, M.E. Newcomer, The structure of human 5-lipoxygenase, Science 331 (2011) 217e219, https://doi.org/10.1126/science.1197203.The. €rving, A.R. Brash, N. Samel, I. Ja €rving, Identification and [12] M. Mortimer, R. Ja characterization of an arachidonate 11R-lipoxygenase, Arch. Biochem. Biophys. 445 (2006) 147e155, https://doi.org/10.1016/j.abb.2005.10.023. €rving, N.C. Gilbert, M.E. Newcomer, N. Samel, Structure of [13] P. Eek, R. J€ arving, I. Ja a calcium-dependent 11R-lipoxygenase suggests a mechanism for Ca2þ regulation, J. Biol. Chem. 287 (2012) 22377e22386, https://doi.org/10.1074/ jbc.M112.343285. €rving, A. Lo ~okene, R. Kurg, L. Siimon, I. Ja €rving, N. Samel, Activation of [14] R. Ja 11R-lipoxygenase is fully Ca2þ-dependent and controlled by the phospholipid composition of the target membrane, Biochemistry 51 (2012) 3310e3320, https://doi.org/10.1021/bi201690z. €tsep, A. Freiberg, I. Ja €rving, N. Samel, A conserved p[15] P. Eek, M.-A. Piht, M. Ra cation and an electrostatic bridge are essential for 11R-lipoxygenase catalysis and structural stability, Biochim. Biophys. Acta 1851 (2015) 1377e1382, https://doi.org/10.1016/j.bbalip.2015.07.007. ~ldemaa, S. Kasvandik, I. Ja €rving, N. Samel, A PDZ-like domain [16] P. Eek, K. Po mediates the dimerization of 11R-lipoxygenase, Biochim. Biophys. Acta 1862 (2017) 1121e1128, https://doi.org/10.1016/j.bbalip.2017.07.012. [17] K.V. Reddy, T. Hammarberg, O. Rådmark, Mg2þ activates 5-lipoxygenase in vitro: dependency on concentrations of phosphatidylcholine and arachidonic acid, Biochemistry 39 (2000) 1840e1848, https://doi.org/10.1021/

85

bi9919246. [18] M. Rakonjac Ryge, M. Tanabe, P. Provost, B. Persson, X. Chen, C.D. Funk, A. Rinaldo-Matthis, B. Hofmann, D. Steinhilber, T. Watanabe, B. Samuelsson, O. Rådmark, A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity, Arch. Biochem. Biophys. 545 (2014) 179e185, https://doi.org/10.1016/j.abb.2014.01.017. [19] A.M. Aleem, J. Jankun, J.D. Dignam, M. Walther, H. Kühn, D.I. Svergun, E. Skrzypczak-Jankun, Human platelet 12-lipoxygenase, new findings about its activity, membrane binding and low-resolution structure, J. Mol. Biol. 376 (2008) 193e209, https://doi.org/10.1016/j.jmb.2007.11.086. [20] G. Bender, E.E. Schexnaydre, R.C. Murphy, C. Uhlson, M.E. Newcomer, Membrane-dependent activities of human 15-LOX-2 and its murine counterpart: implications for murine models of atherosclerosis, J. Biol. Chem. 291 (2016) 19413e19424, https://doi.org/10.1074/jbc.M116.741454. lez-Lafont, [21] A. Di Venere, T. Horn, S. Stehling, G. Mei, L. Masgrau, A. Gonza H. Kühn, I. Ivanov, Role of Arg403 for thermostability and catalytic activity of rabbit 12/15-lipoxygenase, Biochim. Biophys. Acta 1831 (2013) 1079e1088, https://doi.org/10.1016/j.bbalip.2013.02.006. [22] F.W. Studier, Protein production by auto-induction in high density shaking cultures, Protein Expr. Purif. 41 (2005) 207e234. [23] S. Mitra, S.G. Bartlett, M.E. Newcomer, Identification of the substrate access portal of 5-lipoxygenase, Biochemistry 54 (2015) 6333e6342, https://doi.org/ 10.1021/acs.biochem.5b00930. [24] R. Mogul, E. Johansen, T.R. Holman, Oleyl sulfate reveals allosteric inhibition of soybean lipoxygenase-1 and human 15-lipoxygenase, Biochemistry 39 (2000) 4801e4807, https://doi.org/10.1021/bi992805t. [25] A.T. Wecksler, V. Kenyon, J.D. Deschamps, T.R. Holman, Substrate specificity changes for human reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation, Biochemistry 47 (2008) 7364e7375, https://doi.org/ 10.1021/bi800550n. [26] A.T. Wecksler, C. Jacquot, W.A. van der Donk, T.R. Holman, Mechanistic investigations of human reticulocyte 15- and platelet 12-lipoxygenases with arachidonic acid, Biochemistry 48 (2009) 6259e6267, https://doi.org/ 10.1021/bi802332j. [27] A.T. Wecksler, V. Kenyon, N.K. Garcia, J.D. Deschamps, W.A. van der Donk, T.R. Holman, Kinetic and structural investigations of the allosteric site in human epithelial 15-lipoxygenase-2, Biochemistry 48 (2009) 8721e8730, https://doi.org/10.1021/bi9009242. [28] N. Joshi, E.K. Hoobler, S. Perry, G. Diaz, B. Fox, T.R. Holman, Kinetic and structural investigations into the allosteric and pH effect on the substrate specificity of human epithelial 15-lipoxygenase-2, Biochemistry 52 (2013) 8026e8035, https://doi.org/10.1021/bi4010649. [29] H. Kühn, H.-G. Holzhütter, T. Schewe, C. Hiebsch, S.M. Rapoport, The mechanism of inactivation of lipoxygenases by acetylenic fatty acids, Eur. J. Biochem. 139 (1984) 577e583, https://doi.org/10.1111/j.14321033.1984.tb08044.x. [30] S. Peng, W.A. van der Donk, An unusual isotope effect on substrate inhibition in the oxidation of arachidonic acid by lipoxygenase, J. Am. Chem. Soc. 125 (2003) 8988e8989, https://doi.org/10.1021/ja035977t. chal, J.M. Lluch, A. Gonza lez-Lafont, Insights [31] L. Toledo, L. Masgrau, J.-D. Mare into the mechanism of binding of arachidonic acid to mammalian 15lipoxygenases, J. Phys. Chem. B 114 (2010) 7037e7046, https://doi.org/ 10.1021/jp912120n. lez-Lafont, Substrate binding to [32] L. Toledo, L. Masgrau, J.M. Lluch, A. Gonza mammalian 15-lipoxygenase, J. Comput. Aided Mol. Des. 25 (2011) 825e835, https://doi.org/10.1007/s10822-011-9466-5. mez, [33] I. Ivanov, W. Shang, L. Toledo, L. Masgrau, D.I. Svergun, S. Stehling, H. Go lez-Lafont, A. Di Venere, G. Mei, J.M. Lluch, E. Skrzypczak-Jankun, A. Gonza H. Kühn, Ligand-induced formation of transient dimers of mammalian 12/15lipoxygenase: a key to allosteric behavior of this class of enzymes? Proteins Struct. Funct. Bioinfor. 80 (2012) 703e712, https://doi.org/10.1002/ prot.23227. [34] M.C. Reed, A. Lieb, H.F. Nijhout, The biological significance of substrate inhibition: a mechanism with diverse functions, Bioessays 32 (2010) 422e429, https://doi.org/10.1002/bies.200900167.