Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases
2014 (English)In: Archives of Biochemistry and Biophysics, ISSN 0003-9861, E-ISSN 1096-0384, Vol. 555, 9-15 p.Article in journal (Refereed) Published
Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with (FeOH-)-O-III or (MnOH-)-O-III. Hydrogen abstraction by (FeOH-)-O-III 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-H-2(2)]18:2n-6 were almost temperature independent with an apparent D-KIE of similar to 56 at 30 degrees C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8-50 degrees C), and the apparent D-KIE decreased linearly from similar to 38 at 8 degrees C to similar to 20 at 50 degrees 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 0.7 V-gap between the redox potentials of the free metals.
Place, publisher, year, edition, pages
2014. Vol. 555, 9-15 p.
Arrhenius plot, Deuterium kinetic isotope effect, Hydrogen abstraction, Site-directed mutagenesis, Hydrogen tunneling
IdentifiersURN: urn:nbn:se:uu:diva-229705DOI: 10.1016/j.abb.2014.05.014ISI: 000338824100002PubMedID: 24857825OAI: oai:DiVA.org:uu-229705DiVA: diva2:738549