Isotope sensitive branching and kinetic isotope effects in the reaction of deuterated arachidonic acids with human 12- and 15-lipoxygenases.

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.

Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly.

In wild-type soybean LO-1 (WT sLO-1), Asn694 is a weak sixth ligand that is thought to be critical for enzymatic catalysis. In this investigation, N694G sLO-1 was studied to probe its contribution at this sixth ligand position to the kinetic and spectroscopic properties. The k(cat) value of N694G is approximately 230 times lower than that of WT sLO-1 at 25 degrees C, which can be partially explained by a lowered reduction potential of the iron as seen as a shift in the visible ligand-to-metal charge-transfer band (lambda(max) = 410 nm for N694G and lambda(max) = 425 nm for WT sLO-1). This conclusion was supported by a faster rate of oxidation of N694G by the product than that of WT sLO-1 (k(2) = 606 s(-)(1) for N694G and k(2) = 349 s(-)(1) for WT sLO-1). These results suggest a stronger ligand at the active site iron than the native Asn694, which is confirmed to be a water bound to the Fe(II) in the crystal structure. This produces a six-coordinate circular dichroism/magnetic circular dichroism (CD/MCD) spectra for ferrous N694G and an intermediate rhombic electron paramagnetic resonance (EPR) signal for ferric N694G. The EPR spectrum and its pH dependence suggest that the coordination environment of ferric N694G contains one hydroxide and one water. On the basis of both kinetic and structural factors, we propose that the Asn694 water-derived ligand would likely be a hydroxide and the active site, water-derived ligand a water in the ferric state, hence lowering the reaction rate of N694G more than would be expected from the lowered reduction potential alone.

Probing the activity differences of simple and complex brominated aryl compounds against 15-soybean, 15-human, and 12-human lipoxygenase.

Lipoxygenases (LO) have been implicated in asthma, immune disorders, and various cancers. As a consequence of these broad biological implications, there is great interest in understanding the effects of naturally occurring and environmental contaminants against its activity. On the basis of our earlier studies indicating that polybrominated diphenol ethers are potent inhibitors to mammalian 15-LO, we expanded our structure-activity study to include marine-derived brominated phenol ethers (including a newly discovered tribrominated diphenyl ether), dioxins, and bastadins, as well as the synthetic brominated fire retardants, brominated bisphenol A (BBPA), and polybrominated diphenyl ethers (PBDEs). We report herein the effects of 21 simple and complex organobromine compounds against human platelet 12-LO, human reticulocyte 15-LO, and soybean 15-LO-1.

Kinetic investigations of the rate-limiting step in human 12- and 15-lipoxygenase.

Mammalian lipoxygenases have been implicated in several inflammatory disorders; however, the details of the kinetic mechanism are still not well understood. In this paper, human platelet 12-lipoxygenase (12-hLO) and human reticulocyte 15-lipoxygenase-1 (15-hLO) were tested with arachidonic acid (AA) and linoleic acid (LA), respectively, under a variety of changing experimental conditions, such as temperature, dissolved oxygen concentration, and viscosity. The data that are presented show that 12-hLO and 15-hLO have slower rates of product release (k(cat)) than soybean lipoxygenase-1 (sLO-1), but similar or better rates of substrate capture for the fatty acid (k(cat)/K(M)) or molecular oxygen [k(cat)/K(M(O)2)]. The primary, kinetic isotope effect (KIE) for 15-hLO with LA was determined to be temperature-independent and large ((D)k(cat) = 40 +/- 8), over the range of 10-35 degrees C, indicating that C-H bond cleavage is the sole rate-limiting step and proceeds through a tunneling mechanism. The (D)k(cat)/K(M) for 15-hLO, however, was temperature-dependent, consistent with our previous results [Lewis, E. R., Johansen, E., and Holman, T. R. (1999) J. Am. Chem. Soc. 121, 1395-1396], indicating multiple rate-limiting steps. This was confirmed by a temperature-dependent, k(cat)/K(M) solvent isotope effect (SIE), which indicated a hydrogen bond rearrangement step at low temperatures, similar to that of sLO-1 [Glickman, M. H., and Klinman, J. P. (1995) Biochemistry 34, 14077-14092]. The KIE could not be determined for 12-hLO due to its inability to efficiently catalyze LA, but the k(cat)/K(M) SIE was temperature-independent, indicating distinct rate-limiting steps from both 15-hLO and sLO-1.