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  • The reaction of LOX with unlabeled

    2024-06-21

    The reaction of 5-LOX with unlabeled AA generated products from hydrogen abstraction at C7 (95% 5-HETE) with minor products derived from abstraction at C10 (5% 8-HETE). With 7,7--AA the selectivity remained skewed towards C7 hydrogen abstraction (59%) compared to C10 hydrogen abstraction products (41% 8-HETE), but the selectivity was significantly reduced compared to unlabeled AA. The data analysis, after product profiling at substrate concentrations (5–40μM), provided an apparent and / of 20 and 17, respectively, markedly higher than those observed earlier in our study. Thus, our results show that 5-LOX exhibits isotope sensitive branching, however the extent to which the product distribution changed was smaller. An analogous shift in regiospecificity induced by deuterium-labeling has been reported for arachidonic 1,2-Dilauroyl-sn-glycerol in 12-LOX and 15-LOX2. Additionally, oxygenation of 8,11,14-eicosatrienoic acid (lacking the Δ5 double bond) takes place by hydrogen abstraction at C10 and oxygen insertion at C8. The dependence of the steady-state velocity on AA concentration appears to follow Michaelis–Menten kinetics up to 40–50μM (further increase in AA concentration results in significant substrate inhibition), with =2.56±0.25μM/min and =24±6μM. The data presented in this study show that 5-LOX has turnover rates (=0.06s) lower than the other known LOXs. Comparing the catalytic rate () of 5-LOX, when AA or 7,7--AA, were used as substrates, there was ∼7-fold decrease in the rate for 7,7--AA (). Thus, deuterium labeling has dramatic effect on reaction rates, resulting in decreased D-abstraction from C7 as well as slightly faster substrate inhibition. A similar trend was observed when the reactions were separately analyzed for 5-HETE and 8-HETE formation with more pronounced decrease in the rate of 5-HETE formation in case of labeled substrate (, ). There is only a small difference between the KIE observed for (20±4) and / (17±2). A possible reason for this observation can be that 5-LOX displays strong substrate inhibition at higher substrate concentrations, which could reduce the KIE, indicating that it would be less dependent on the isotopic label and more dependent on the functional integrity of the enzyme. Results from viscosity experiments (explained below) show that the steps after the chemical step affect the to a larger extent than /. However, the KIE for 5-LOX that will describe its catalytic mechanism most ‘truly’ will be through the / value since that value originates from measurements done at low AA concentrations where the enzyme is less affected by substrate inhibition. Also, we could see only a 2-fold change in (/=2.18) upon isotope labeling, thus we speculate that KIE for / is not strongly affected by substrate binding at low substrate concentrations. The overall structural similarity between the substrate and product, further envisage us to rule out any major contribution provided by the product release. Thus, we suggest that the KIE observed for the / is mainly dictated by the chemical step when hydrogen is abstracted. Enzymes displaying moderate to high KIEs have been described to use tunneling as a mechanism for hydrogen transfer. The extent of tunneling is related to the degree of dependency of rates on variable temperatures., To investigate the nature of the rate-determining step in catalysis, temperature dependence of initial rates for the reaction of 5-LOX with AA and 7,7--AA was determined. In this study we compared the rates at temperatures ranging from 4°C to 35°C using 30μM substrate, a concentration that was chosen to avoid substrate inhibition. The temperature dependence of the rates () for both hydrogen and deuterium atom abstraction were fitted with the empirical Arrhenius equation =e−), where represents the gas constant, is the absolute temperature, is the activation energy, and is the Arrhenius prefactor (). The Arrhenius plot analysis shows that the reaction occurs even at lower temperatures and there is linear dependence for both AA as well 7,7-AA, AA being more flat than that for -AA. The calculated Arrhenius prefactor ratio (/=0.21) is low, associated with a very small difference in activation energy for the two substrates ((D)−(H)=3.6J/mol). All these factors along with similar geometries of reactant and the product suggest that there is a possibility of tunneling involved as a mechanism during H-transfer. However the data shows slight temperature dependence for (C–H), contrasting with either semi-classical predictions or the expected behavior for tunneling correction models., , These results cannot be attributed to kinetic complexity, since the KIEs shown are computed for a single C–H bond cleavage step. Thus, further analysis is required to confirm the mechanism of H-transfer, which is beyond the scope of this study.