Journal
BIOCHEMISTRY
Volume 48, Issue 26, Pages 6259-6267Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi802332j
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Funding
- National Institutes of Health [S10-RR20939, GM44911, GM56062]
- American Heart Association predoctoral fellowship [0615604Z]
- California Institute for Quantitative Biosciences
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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 atorn 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 mu M but increases the K-M[O-2] for linoleic acid (LA) to 22 mu M, such that the k(cat)/K-M[O-2] values become similar for both substrates (similar to 0.3 s(-1) mu M-1). Considering that the oxygen concentration in cancerous tissue can be less than 5 mu M, this result may have cellular implications with respect to the substrate specificity of 15-hLO-1.
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