Kinetic investigation of human 5-lipoxygenase with arachidonic acid
2016 (English)In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 26, no 15, 3547-3551 p.Article in journal (Refereed) Published
Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A(4), a pivotal intermediate in the biosynthesis of the leukotrienes, a family of proinflammatory lipid mediators. 5-LOX has thus gained attention as a potential drug target. However, details of the kinetic mechanism of 5-LOX are still obscure. In this Letter, we investigated the kinetic isotope effect (KIE) of 5-LOX with its physiological substrate, arachidonic acid (AA). The observed KIE is 20 +/- 4 on k(cat) and 17 +/- 2 on k(cat)/K-M at 25 degrees C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35 degrees C. Also, we observed low Arrhenius prefactor ratio (A(H)/A(D) = 0.21) and a small change in activation energy (E-a(D) - E-a(H) = 3.6 J/mol) which suggests that 5-LOX catalysis involves tunneling as a mechanism of H-transfer. The measured KIE for 5-LOX involves a change in regioselectivity in response to deuteration at position C7, resulting in H-abstraction form C10 and formation of 8-HETE. The viscosity experiments influence the (H)k(cat), but not (D)k(cat). However the overall kcat/K-M is not affected for labeled or unlabeled AA, suggesting that either the product release or conformational rearrangement might be involved in dictating kinetics of 5-LOX at saturating conditions. Investigation of available crystal structures suggests the role of active site residues (F421, Q363 and L368) in regulating the donor-acceptor distances, thus affecting H-transfer as well as regiospecificity. In summary, our study shows that that the H-abstraction is the rate limiting step for 5-LOX and that the observed KIE of 5-LOX is masked by a change in regioselectivity.
Place, publisher, year, edition, pages
Elsevier, 2016. Vol. 26, no 15, 3547-3551 p.
Lipoxygenase, Kinetic isotope effect, Arachidonic acid
IdentifiersURN: urn:nbn:se:kth:diva-192401DOI: 10.1016/j.bmcl.2016.06.025ISI: 000380574100030PubMedID: 27363940ScopusID: 2-s2.0-84977613910OAI: oai:DiVA.org:kth-192401DiVA: diva2:970719
FunderSwedish Research Council
QC 201609142016-09-142016-09-122016-09-14Bibliographically approved