Elsevier

Bioorganic & Medicinal Chemistry

Volume 17, Issue 18, 15 September 2009, Pages 6534-6539
Bioorganic & Medicinal Chemistry

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1

https://doi.org/10.1016/j.bmc.2009.08.005Get rights and content

Abstract

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.

Introduction

Lipoxygenases (LO) are a family of metallo-enzymes which initiate oxylipin signalling cascades in response to metabolic needs and external stimuli. Signal initiation begins with the catalytic incorporation of molecular oxygen into unsaturated fatty acids, producing the respective hydroperoxide fatty acid products.1 There are three human LO (hLO) isozymes of pharmacological importance (5-hLO, 12-hLO and 15-hLO) which are designated by their relative oxidation position on arachidonic acid (AA).2 The hydroperoxide products (hydroperoxyeicosatetraenoic acids (HPETEs)) regulate pro-inflammatory (leukotrienes) and anti-inflammatory/resolution (lipoxins and resolvins) responses.3 The LO metabolites of AA, as well as linoleic acid (LA), have been implicated in a variety of inflammatory diseases and cancers, making hLO a possible target for drug therapy.4

Our current understanding of LO biochemistry comes from extensive kinetic, structural and mechanistic investigations of the soybean lipoxygenase-1 (sLO-1).5, 6, 7, 8 In plants, lipoxygenases react with the C18 polyunsaturated fatty acids, LA and α-linolenic acid (ALA), producing predominately 13-hydroperoxyoctadecadienoic acid (13-HPODE) and 13-hydroperoxyoctadecatrienoic acid (13-HPOTrE), respectively.9 The sLO-1 metabolites of these two fatty acids have many physiological effects, including the regulation of germination and senescence; with one of the most, well-characterized metabolic pathways being that of jasmonic acid synthesis, a powerful biomolecule used for plant defense against pathogens.10, 11 sLO-1 has been consistently used as a model for 15-hLO-1 due to their mechanistic similarities in AA metabolism, both producing 15-HPETE, as well as their structural similarities in both the alpha-helical (catalytic) and beta-barrel (membrane associated) domains.5, 8 In addition, both sLO-1 and 15-hLO-1 react preferentially towards AA over LA, adding substrate specificity to the list of similarities between these two LOs.12, 13

With regards to 15-hLO, there is growing evidence that substrate specificity may be the underlying cause for the advancement of certain diseases.14, 15, 16, 17 For example, reticulocyte 15-hLO-1 reacts preferentially with LA to produce 13-HPODE, which causes prostate carcinoma cells to undergo proliferation and differentiation, while epithelial 15-hLO-2 reacts preferentially with AA to produce 15-HPETE, which inhibits cell proliferation.18, 19 Therefore, it is proposed that 15-hLO-1 and its product, 13-HPODE, promote cancer progression, while 15-hLO-2 and its product, 15-HPETE, inhibit cancer progression. This hypothesis was recently supported by the fact that the substrate specificity of the 15-hLO isozymes is directly affected by an allosteric product-feedback mechanism,12 which could modify the ratio of LO products in the cell, and affect its carcinogenic progression. This result of 15-hLO-1 raised the question of whether LO products affected the substrate specificity of sLO-1 as well, since sLO-1 is similar in many respects to 15-hLO-1.

In the current work, we have investigated the allosteric effect of the reduced LO products, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), 13-(S)-hydroxyoctadecatrienoic acid (13-HOTrE) and 12-(S)-hydroxyeicosatetraenoic acid (12-HETE), on sLO-1 substrate specificity with the endogenous substrate mixture, ALA:LA, and the non-endogenous substrate mixture, AA:LA. These results demonstrate that there is no observed allosteric product feedback with sLO-1’s endogenous products, however, the non-endogenous product, 12-HETE, increased the substrate specificity of sLO-1 towards AA, when challenged with an LA/AA mixture. Allosteric effects on substrate specificity of sLO-1 were also probed with three well-characterized inhibitors of sLO-1 (Fig. 1), oleic acid (OA, competitive inhibition), oleyl sulfate (OS, allosteric inhibition) and palmitoleyl sulfate (PS, mixed-type inhibition) on the substrate specificity of sLO-1. Intriguingly, the inhibitors which bind the allosteric site (OS and PS) displayed an effect on the substrate specificity of sLO-1, correlating to their respective binding affinities towards the allosteric site,20, 21, 22, 23 while the competitive inhibitor, OA, had no effect. These results may have implications in the potential targeting of 15-hLO in human disease.

Section snippets

Materials

All commercial fatty acids (Sigma–Aldrich Chemical Company) were re-purified using a Higgins HAIsil Semi-Preparative (5 μm, 250 × 10 mm) C-18 column. Solution A was 99.9% MeOH and 0.1% acetic acid; solution B was 99.9% H2O and 0.1% acetic acid. An isocratic elution of 85% A:15% B was used to purify all fatty acids, which were stored at −80 °C for a maximum of 6 months. LO products were generated by reacting substrate with the appropriate LO isozyme (13-HPODE from sLO-1 and LA, 13-HPOTrE from sLO-1

Determination of substrate specificity using the competitive substrate capture method with substrate mixtures of arachidonic acid:linoleic acid, and α-linolenic acid:linoleic acid

The ratio of substrate turnover for sLO-1 using either AA:LA or ALA:LA reaction mixtures (100 mM Borate, pH 9.2, 22 °C) were determined by monitoring the simultaneous product formation using the HPLC method described above. The competitive substrate capture (kcat/KM)AA/(kcat/KM)LA ratio was determined to be 1.8 ± 0.2, in agreement with the steady-state kinetic data and previously published results.12, 13 The (kcat/KM)ALA/(kcat/KM)LA ratio was determined to be 0.75 ± 0.05, demonstrating sLO-1 displays

Discussion

In our previous report, we uncovered an allosteric product-feedback mechanism that directly affected the substrate specificity of 15-hLO-1.12 In order to probe if the substrate specificity of sLO-1 can also be manipulated by allosteric product binding, we investigated the effect of the addition of 13-HODE, 13-HOTrE and 12-HETE, on the substrate specificity for the endogenous sLO-1 substrate mixture, ALA:LA, and for the non-endogenous substrate mixture, AA:LA. The results from these experiments

Acknowledgements

The authors acknowledge the Holman group members for helpful discussions and proof reading the manuscript.

References and notes (29)

  • E.I. Solomon et al.

    Chem. Biol.

    (1997)
  • S. Yamamoto

    Biochim. Biophys. Acta

    (1992)
  • E.H. Oliw

    Prostaglandins Other Lipid Mediat.

    (2002)
  • A. Grechkin

    Prog. Lipid Res.

    (1998)
  • L.C. Hsi et al.

    J. Biol. Chem.

    (2002)
  • L.C. Hsi et al.

    J. Biol. Chem.

    (2001)
  • S.B. Shappell et al.

    Neoplasia

    (2001)
  • J.D. Deschamps et al.

    Bioorg. Med. Chem.

    (2006)
  • B. Samuelsson et al.

    Science

    (1987)
  • V.E. Steele et al.

    Cancer Epidem. Biomar. Prevent.

    (1999)
  • S.T. Prigge et al.

    Proteins

    (1996)
  • M.H. Glickman et al.

    Biochemistry

    (1995)
  • M.J. Knapp et al.

    Biochemistry

    (2003)
  • W. Minor et al.

    Biochemistry

    (1996)
  • Cited by (0)

    This work was supported by the National Institutes of Health (GM56062, TRH).

    View full text