title: Kinetic investigation of human 5-lipoxygenase with arachidonic acid.
abstract: Human 5-lipoxygenase (5-LOX) is responsible for the formation of leukotriene (LT)A4, 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 kcat and 17±2 on kcat/KM at 25°C indicating a non-classical reaction mechanism. The observed rates show slight temperature dependence at ambient temperatures ranging from 4 to 35°C. Also, we observed low Arrhenius prefactor ratio (AH/AD=0.21) and a small change in activation energy (Ea(D)-Ea(H)=3.6J/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)kcat, but not (D)kcat. However the overall kcat/KM 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.
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: Monica
authors.initials: M
authors.lastname: Mittal
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden; School of Technology and Health, KTH Royal Institute of Technology, 14183 Huddinge, Sweden.
authors.forename: Ramakrishnan B
authors.initials: RB
authors.lastname: Kumar
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: Navisraj
authors.initials: N
authors.lastname: Balagunaseelan
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.affiliation: Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden.
authors.forename: Caroline
authors.initials: C
authors.lastname: Jegerschöld
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: Olof
authors.initials: O
authors.lastname: Rådmark
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden.
authors.forename: Jesper Z
authors.initials: JZ
authors.lastname: Haeggström
authors.affiliation: Division of Physiological Chemistry II, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden. Electronic address: Agnes.Rinaldo-Matthis@ki.se.
authors.forename: Agnes
authors.initials: A
authors.lastname: Rinaldo-Matthis
mesh_terms: D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D016718:Arachidonic Acid / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D016859:Lipoxygenase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D015394:Molecular Structure; D013329:Structure-Activity Relationship
chemical_list: D016859:Lipoxygenase Inhibitors; D016718:Arachidonic Acid; D001094:Arachidonate 5-Lipoxygenase
keywords: Arachidonic acid; Kinetic isotope effect; Lipoxygenase