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  • br Acknowledgments This work was supported by

    2022-11-09


    Acknowledgments This work was supported by a grant of the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project no. PN-II-ID-PCE-2011-3-0571, awarded to F.A.M. D.V.N. was co-financed from the European Social Fund through Sectorial Operational Program Human Resources Development 2007–2013, project no. POSDRU/CPP107/DMI 1.5/S/77082, “Doctoral Scholarships for eco-economy and bio-economic complex training to ensure the food and feed safety and security of anthropogenic ecosystems". M.S. was supported by the strategic Grant POSDRU/159/1.5/S/133391, Project “Doctoral and Post-doctoral programs of excellence for highly qualified human resources training for research in the field of Life sciences, Environment and Earth Science” co-financed by the European Social Fund within the Sectorial Operational Program Human Resources Development 2007–2013. The funding sources had no involvement in the collection, analysis, interpretation of data, writing of the report, and in the decision to submit the article for publication.
    Lipoxygenases are non-heme iron-containing enzymes, present in bacteria, plants as well as mammals catalyzing the stereospecific and regiospecific peroxidation of natural polyunsaturated fatty acids to hydroperoxy derivatives., The products of lipoxygenase reactions are involved in a variety of biological functions in almost all phyla such as signaling, germination and senescence., Human 5-LOX is implicated in a variety of diseases ranging from asthma to cancer, thus gaining importance as a potential therapeutic target, , and emphasizing the need for further investigation of its catalytic mechanism. 5-LOX catalyzes a 2-step reaction where arachidonic pilocarpine hydrochloride australia (AA) is oxygenated, resulting in formation of 5()-hydroperoxy-6(),8(),11(),14()-eicosatetraenoic acid (5-HPETE), with subsequent formation of leukotriene A (LTA). During the first step, 5-LOX binds its substrate AA and abstracts the hydrogen, H, from position C7. After radical rearrangement, O is inserted at position C5 resulting in the formation of 5(S)-HPETE. In the second step, abstraction of the hydrogen, H, from position C10 generates the epoxide LTA (). Isotope-labeling studies provides a handle to investigate the mechanism of action of various enzymes. Lipoxygenases, especially soybean LOX, have attracted considerable attention due to the large kinetic isotope effects (KIEs) exhibited in reactions with linoleic acid (LA). However, there is dearth of studies investigating the mechanism of action of lipoxygenases with arachidonic acid as substrate. To investigate the catalytic mechanism of human 5-LOX, we used arachidonic acid (AA) and 7,7--arachidonic acid (7,7-AA) as substrates. Although the double label of 7,7--arachidonic acid results in a combination of primary and secondary KIEs, we decided to prepare this compound to avoid any complications from a potential change in stereoselectivity in the hydrogen atom abstraction step, as has been reported in earlier studies. The reaction of AA and 7,7--AA with 5-LOX was monitored at 235nm for the formation of HETEs as shown in Figure S1. 13-HPODE was added to oxidize Fe to Fe, thus reducing the lag phase of the reaction. By fitting the Michaelis–Menten rate equation to the data, the kinetic parameters shown in and were obtained. The apparent primary kinetic isotope effect on and / at 25°C was 6.0 and 2.3, respectively when comparing AA and 7,7--AA kinetics. These values are smaller than the previously reported numbers under similar conditions for the oxidation of perdeuterated LA by lipoxygenases, which include a of 80 and 48 for soybean LOXs and 15-LOX-1, respectively, but 10 for 15-LOX-1 and AA., , As mentioned in previous reports, one possible mechanism resulting in lower pilocarpine hydrochloride australia isotope effects involves a change in regioselectivity in response to deuteration. To investigate a possible shift in regioselectivity, the product composition of the reactions of 5-LOX with arachidonic acid and its deuterated analog (5–30μM), were determined by HPLC after quenching the reactions with methanol. To quantify the analysis, 13-HPODE was added to the kinetic assays as described above and the product distribution was determined. The representative chromatograms for the reactions of 5-LOX with 30μM of AA and 7,7--AA, are shown in Figure S3. We observed a significant reduction in 5-HETE formation, along with upsurge in rate of 8-HETE formation (A and B).