Kinetic and Structural Investigations of the Allosteric Site in Human Epithelial 15-Lipoxygenase-2

Abstract

Allosteric regulation of human lipoxygenase (hLO) activity has recently been implicated in the cellular biology of prostate cancer. In the current work, we present isotope effect, pH and substrate inhibitor data of epithelial 15-hLO-2, which probe the allosteric effects on its mechanistic behavior. The Dkcat/KM for 15-hLO-2, with AA and LA as substrate, is large indicating hydrogen atom abstraction is the principle rate-determining step, involving a tunneling mechanism for both substrates. For AA, there are multiple rate determining steps (RDS) at both high and low temperature, with both diffusion and hydrogen bonding rearrangements contributing at high temperature, but only hydrogen bonding rearrangements contributing at low temperature. The observed kinetic dependency on the hydrogen bonding rearrangement is eliminated upon addition of the allosteric effector, 13-(S)-hydroxyoctadecadienoic acid (13-HODE), however, no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (kcat/KM)AA/(kcat/KM)LA ratio was observed to have a pH dependence, which was fit with a titration curve (pKa = 7.7), suggesting the protonation of a histidine residue, which could hydrogen bond with the carboxylate of 13-HODE. Assuming this interaction, 13-HODE was docked to the solvent exposed histidines of a 15-hLO-2 homology model and found to bind well with H627, suggesting a potential location for the allosteric site. Utilizing d31-LA as an inhibitor, it was demonstrated that the binding of d31-LA to the allosteric site changes the conformation of 15-hLO-2 such that the affinity for substrate increases. This result suggests that allosteric binding locks the enzyme into a catalytically competent state, which facilitates binding of LA and decreases the (kcat/KM)AA/(kcat/KM)LA ratio. Finally, the magnitude of the 13-HODE KD for 15-hLO-2 is over 200-fold lower than that of 13-HODE for 15-hLO-1, changing the substrate specificity of 15-hLO-2 to 1.9, which would alter the LO product distribution by increasing the production of the pro-tumorigenic 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.

Dimerization of human 5-lipoxygenase

“…Though, different reaction kinetics for both quaternary structures are conceivable, similarly to what was already discussed for 12-LO (Aleem et al , 2008 ) and 15-LO2 (Wecksler et al , 2009 ). An impact of dimerization on the reaction kinetics is presumable due to the observation that the predicted dimer interface is located at the proposed substrate and oxygen entry site of LOs (Ivanov et al , 2010 ).…”

Effect of soluble epoxide hydrolase polymorphism on substrate and inhibitor selectivity and dimer formation

“…The substrate preference obtained for the WT is similar to the one calculated from the kinetic constants ( 18 ), underlining the accuracy of the method used. It also indicates that there are probably no substrate/product allosteric interactions as seen with lipoxygenase ( 29 ). While the total amount of diol formed by each SNP was different, the relative amounts of diol among the different EpFA substrates were similar (supplementary Fig.…”

Rationally Designed Multitarget Agents Against Inflammation and Pain

“…It is unclear if these responses are due to effects on the transcriptional regulation of the associated enzymes, or if this is a result from direct effects on enzyme activity. However, it has been shown that LOX metabolites of ARA and LA can differentially affect the substrate selectivity [129] and reaction mechanism [130] of the LOX enzymes through allosteric regulation. This suggests a direct protein/metabolite interaction may regulate crosstalk within the ARA cascade, and that metabolite interactions may extend into other pathways such LA and ω-3 fatty acids metabolizing cascades.…”