Isotope Sensitive Branching and Kinetic Isotope Effects in the Reaction of Deuterated Arachidonic Acids with Human 12- and 15-Lipoxygenases

Abstract

Lipoxygenases (LOs) catalyze lipid peroxidation and have been implicated in a number of human diseases connected to oxidative stress and inflammation. These enzymes have also attracted considerable attention due to large kinetic isotope effects (30−80) for the rate-limiting hydrogen abstraction step with linoleic acid (LA) as substrate. Herein, we report kinetic isotope effects (KIEs) in the reactions of three human LOs (platelet 12-hLO, reticulocyte 15-hLO-1, and epithelial 15-hLO-2) with arachidonic acid (AA). Surprisingly, the observed KIEs with AA were much smaller than the previously reported values with LA. Investigation into the origins for the smaller KIEs led to the discovery of isotope sensitive branching of the reaction pathways. Product distribution analysis demonstrated an inversion in the regioselectivity of 15-hLO-1, with hydrogen abstraction from C13 being the major pathway with unlabeled AA, but abstraction from C10 predominating when the methylene group at position 13 was deuterated. Smaller but clear changes in regioselectivity were also observed for 12-hLO and 15-hLO-2. Keywordsisotope sensitive branching; lipoxygenase; arachidonic acid Lipoxygenases (LOs) are non-heme iron proteins that catalyze the hydrogen atom abstraction from a bisallylic position in polyunsaturated fatty acids and the subsequent addition of molecular oxygen to the resulting substrate radical to generate hydroperoxide products (1). Mammalian LOs catalyze key steps in the conversion of arachidonic acid (AA), 1 a C20 tetraunsaturated fatty acid (20:4, ω−6), to lipoxins and leukotrienes (2). They have been implicated in a number of pathologies including cancer (3,4), atherosclerosis (5) and Alzheimer's disease (6,7). In plants, LOs convert linoleic acid (LA), a C18 bisunsaturated fatty acid (18:2, ω−6), into 13-hydroperoxyoctadecadienoic acid (13-HPODE), a precursor for † This work was supported by the National Institutes of Health (GM44911, WAV and GM56062, TRH) and American Heart Association pre-doctoral fellowships (0615604Z, CJ and 0310006Z, CMM).vddonk@uiuc.edu. tholman@chemistry.ucsc.edu. Supporting information Synthetic procedures for 10,10,13,13-d 4 arachidonic acid and HPLC traces displaying product distributions of the human lipoxygenase reactions are available free of charge via the internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 July 8. Published in final edited form as:Biochemistry. jasmonates and aldehydes involved in signaling, germination, and senescence (8). In mammals, the isozymes are named according to the position of AA that reacts with molecular oxygen (Figure 1). Several human LOs (5-, 12-, and 15-hLOs) have thus far been identified (9), with this study focusing on the latter two groups. Two isozymes of 15-hLO have been characterized in humans and are called 15-hLO-1 (also termed 12/15-hLO) (10,11) and 15-hLO-2 (12). The former exhibits a product selectivity of 15-HPETE over 12-HPETE of about a 9:1 (13), whereas the lat...

Influence of Substrate Dideuteration on the Reaction of the Bifunctional Heme Enzyme Psi Factor Producing Oxygenase A (PpoA)

“…[18] A representative chromatogram of the LC/MS analysis as well as the corresponding MS/MS spectra are provided in the Supporting Information. The obtained spectra are in accordance with spectra reported previously.…”

Substrate binding to mammalian 15-lipoxygenase

“…Therefore, they are of interest as targets in drug design. Revealing the exact role of LOs in human disease is hampered by an incomplete understanding of LOs activity, including substrate specificity, substrate alignment in the active site and allosteric regulation [2,3]. Recently, the substrate specificity (arachidonic acid, AA vs. linoleic acid, LA) of the human 15-hLO isozymes has been suggested to be relevant in cancer progression [4].…”

“…Recently, the substrate specificity (arachidonic acid, AA vs. linoleic acid, LA) of the human 15-hLO isozymes has been suggested to be relevant in cancer progression [4]. Significant differences between reaction of 15-hLO with AA or LA have also been reported by the measured kinetic isotope effects (KIEs) on k cat and k cat /K M [3,4]. The generally accepted catalytic reaction for this enzyme (Scheme 1) involves a hydrogen transfer that is thought to be the rate-determining (RDS) step.…”

Interpretation of Kinetic Isotope Effects in Enzymatic Cleavage of Carbon-Hydrogen Bonds

“…It seems likely that these low values are due to contamination of the corresponding rates by isotope-independent slow steps in the reaction sequence. Such contaminations have indeed been observed in other reactions catalyzed by lipoxygenase [16]. It is associated with fast tunneling steps, which appear to have evolved so as to reach a rate approaching that of other steps in the reaction.…”