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 Dk(cat)/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 temperatures, 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), and no allosteric effects were seen on diffusion or hydrogen atom abstraction. The (k(cat)/KM)AA/(k(cat)/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 (k(cat)/KM)AA/(k(cat)/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. This would alter the LO product distribution and increase the production of the pro-tumorigenic, 13-HODE, possibly representing a pro-tumorigenic feedback loop for 13-HODE and 15-hLO-2.

Figure 1

pH dependence of k_cat/K_M (open squares) and k_cat (closed circles) for 15-hLO-2 with AA. Enzymatic reactions were performed in 25 mM HEPES buffer at 22 °C.

Figure 2

Temperature dependence of k_cat/K_M (open squares) and k_cat (closed circles) for 15-hLO-2 with AA. Enzymatic reactions were performed in 25 mM HEPES buffer at pH 7.5.

Figure 3

Temperature dependence of competitive KIE for 15-hLO-2: ^Dk_cat/K_M[AA] (open circle) and ^Dk_cat/K_M[LA] (closed square). Enzymatic assays were performed at 5 μM substrate concentrations in 25 mM HEPES Buffer (pH 7.5).

Figure 4

Effect of relative viscosity (η/η⁰) on normalized values of reciprocal k_cat/K_M[AA] at 37 °C. The slope of the line is 0.50 (± 0.03) and -0.062 (± 0.004) for k_cat/K_M (open circles) and k_cat (close circles), respectively. Solid line is the theoretical behavior for a fully-diffusion controlled reaction. Enzymatic assays were performed in 25 mM HEPES Buffer (pH 7.5) with 0%, 21.6% and 30% w/v Glucose.

Figure 5

Temperature dependence of the SIE for 15-hLO-2 with AA as substrate: k_cat (close squares) and k_cat/K_M (open circles). Enzymatic assays were performed in 25 mM HEPES buffer (pH 7.5).

Figure 6

pH dependence of the substrate specificity for 15-hLO-2, using the competitive substrate capture. Enzymatic assays were performed at 1 μM substrate concentrations in 50 mM MES (pH 6-7), 50 mM HEPES (pH 7-8.5), 50 mM CHES (pH8.5-10) at 37 °C and constant ionic strength (200 mM). Fitting the data revealed a pKa_(app) = 7.7 ± 0.1.

Figure 7

Figure 7A. 15-hLO-2 homology model docked with 13-HODE (space filling model) in the proposed allosteric site. Figure generated with Pymol. Figure 7B. Magnified view of 15-hLO-2 homology model docked with 13-HODE (stick model) in the proposed allosteric site, depicting hydrogen bonding interactions of the carboxylate of 13-HODE with H627 and its alcohol moiety with R407 and Y408. Figure generated with Pymol.

Figure 7

Figure 7A. 15-hLO-2 homology model docked with 13-HODE (space filling model) in the proposed allosteric site. Figure generated with Pymol. Figure 7B. Magnified view of 15-hLO-2 homology model docked with 13-HODE (stick model) in the proposed allosteric site, depicting hydrogen bonding interactions of the carboxylate of 13-HODE with H627 and its alcohol moiety with R407 and Y408. Figure generated with Pymol.

Figure 8

Steady-state inhibition kinetics data for the determination of K_i and K_i′ for 15-hLO-2 with d₃₁-LA as an inhibitor, with and without 13-HODE addition. (A) K_M(app)/k_cat(i) (slope) versus [d₃₁-LA] μM is the secondary re-plot of inhibition data used to determine K_i. Without 13-HODE addition K_i = 17 ± 3 μM (open circles) and with 13-HODE addition (1 μM) K_i = 5 ± 1 μM (closed squares). (B) 1/k_cat(i) (y-intercept) versus d₃₁-LA μM is a secondary re-plot of inhibition data used to determine K_i′. Without 13-HODE addition K_i′ = 149 ± 10 μM (open circles) and with 13-HODE addition K_i′ is not observed (closed squares). Enzymatic assays were performed in 25 mM HEPES (pH 7.5) at 22 °C with AA as substrate.