title: Mechanistic investigations of human reticulocyte 15- and platelet 12-lipoxygenases with arachidonic acid.
abstract: Human reticulocyte 15-lipoxygenase-1 (15-hLO-1) and human platelet 12-lipoxygenase (12-hLO) have been implicated in a number of diseases, with differences in their relative activity potentially playing a central role. In this work, we characterize the catalytic mechanism of these two enzymes with arachidonic acid (AA) as the substrate. Using variable-temperature kinetic isotope effects (KIE) and solvent isotope effects (SIE), we demonstrate that both k(cat)/K(M) and k(cat) for 15-hLO-1 and 12-hLO involve multiple rate-limiting steps that include a solvent-dependent step and hydrogen atom abstraction. A relatively low k(cat)/K(M) KIE of 8 was determined for 15-hLO-1, which increases to 18 upon the addition of the allosteric effector molecule, 12-hydroxyeicosatetraenoic acid (12-HETE), indicating a tunneling mechanism. Furthermore, the addition of 12-HETE lowers the observed k(cat)/K(M) SIE from 2.2 to 1.4, indicating that the rate-limiting contribution from a solvent sensitive step in the reaction mechanism of 15-hLO-1 has decreased, with a concomitant increase in the C-H bond abstraction contribution. Finally, the allosteric binding of 12-HETE to 15-hLO-1 decreases the K(M)[O(2)] for AA to 15 microM but increases the K(M)[O(2)] for linoleic acid (LA) to 22 microM, such that the k(cat)/K(M)[O(2)] values become similar for both substrates (approximately 0.3 s(-1) microM(-1)). Considering that the oxygen concentration in cancerous tissue can be less than 5 microM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, California 95064, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Wecksler
authors.forename: Cyril
authors.initials: C
authors.lastname: Jacquot
authors.forename: Wilfred A
authors.initials: WA
authors.lastname: van der Donk
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000737:chemistry; D000494:Allosteric Regulation; D001092:Arachidonate 12-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000235:genetics / Q000378:metabolism*; D016718:Arachidonic Acid / Q000378:metabolism*; D055162:Biocatalysis; D001792:Blood Platelets / Q000201:enzymology; D002247:Carbon Isotopes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D015289:Leukotrienes / Q000737:chemistry; D019787:Linoleic Acid / Q000737:chemistry; D008041:Linoleic Acids / Q000737:chemistry; D008054:Lipid Peroxides / Q000737:chemistry; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012156:Reticulocytes / Q000201:enzymology; D012997:Solvents / Q000737:chemistry; D013696:Temperature
chemical_list: D002247:Carbon Isotopes; D015289:Leukotrienes; D008041:Linoleic Acids; D008054:Lipid Peroxides; D011994:Recombinant Proteins; D012997:Solvents; C038649:13-hydroperoxy-9,11-octadecadienoic acid; D016718:Arachidonic Acid; C024348:13-hydroxy-9,11-octadecadienoic acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; C021201:12-HPETE; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; D019787:Linoleic Acid; D001092:Arachidonate 12-Lipoxygenase; C476558:ALOX12 protein, human; C515716:ALOX15 protein, human; D001093:Arachidonate 15-Lipoxygenase; D010100:Oxygen