title: Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases.
abstract: Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with FeIIIOH- or MnIIIOH-. Hydrogen abstraction by FeIIIOH- of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-2H2]18:2n-6 were almost temperature independent with an apparent D-KIE of ∼56 at 30°C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8-50°C), and the apparent D-KIE decreased linearly from ∼38 at 8°C to ∼20 at 50°C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between 13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor-acceptor distances, and to the metal ligands, which may not equalize the 0.7V-gap between the redox potentials of the free metals.
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Anneli
authors.initials: A
authors.lastname: Wennman
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
authors.forename: Saeid
authors.initials: S
authors.lastname: Karkehabadi
authors.affiliation: Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden. Electronic address: Ernst.Oliw@farmbio.uu.se.
authors.forename: Ernst H
authors.initials: EH
authors.lastname: Oliw
mesh_terms: D001093:Arachidonate 15-Lipoxygenase / Q000737:chemistry; D055162:Biocatalysis; D003903:Deuterium; D005231:Fatty Acids, Unsaturated / Q000737:chemistry; D006801:Humans; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D019787:Linoleic Acid / Q000737:chemistry; D008084:Lipoxygenase / Q000737:chemistry* / Q000235:genetics; D008345:Manganese / Q000737:chemistry*; D009154:Mutation; D010940:Plant Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013025:Glycine max; D013696:Temperature; D013816:Thermodynamics; D017962:alpha-Linolenic Acid / Q000737:chemistry
chemical_list: D005231:Fatty Acids, Unsaturated; D010940:Plant Proteins; D011994:Recombinant Proteins; D017962:alpha-Linolenic Acid; D008345:Manganese; D019787:Linoleic Acid; D003903:Deuterium; D007501:Iron; C101084:lipoxygenase L-1; D008084:Lipoxygenase; C515716:ALOX15 protein, human; D001093:Arachidonate 15-Lipoxygenase
keywords: Arrhenius plot; Deuterium kinetic isotope effect; Hydrogen abstraction; Hydrogen tunneling; Site-directed mutagenesis