Mechanistic investigations of human reticulocyte 15- and platelet 12-lipoxygenases with arachidonic acid

Aaron T. Wecksler, Cyril Jacquot, Wilfred A. Van Der Donk, Theodore R. Holman

Research output: Contribution to journalArticlepeer-review

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 kcat/KM and kcat 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 kcat/KM 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 kcat/KM 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 KM[O2] for AA to 15 μM but increases the KM[O2] for linoleic acid (LA) to 22 μM, such that the kcat/KM[O2] values become similar for both substrates (∼0.3 s-1 μM-1). Considering that the oxygen concentration in cancerous tissue can be less than 5 μM, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.

Original languageEnglish (US)
Pages (from-to)6259-6267
Number of pages9
JournalBiochemistry
Volume48
Issue number26
DOIs
StatePublished - Jul 7 2009

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Mechanistic investigations of human reticulocyte 15- and platelet 12-lipoxygenases with arachidonic acid'. Together they form a unique fingerprint.

Cite this