Substrate specificity changes for human reticulocyte and epithelial 15-lipoxygenases reveal allosteric product regulation

Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In this paper, we present a novel method for accurately measuring the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the (k(cat)/K-m)(AA)/(k(cat)/K-m)(LA) ratio more than 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-(S)-hydroperoxyeicosatetraenoic acid (12-HPETE), affects only the ratio of 15-hLO-1 (more than 5-fold). In addition, the reduced products, 13-(S)-hydroxyoctadecadienoic acid (13-HODE) and 12-(S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the (k(cat)/K-m)(AA)/(k(cat)/K-m)(LA) ratio. These results, coupled with the dependence of the 15-hLO-1 k(cat)/K-m kinetic isotope effect ((D)k(cat)/K-m) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., and Holman, T. R. (1999) Biochemistry 39, 4801-4807], is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may be relevant with respect to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell.