Modulation of Human 5-Lipoxygenase Activity by Membrane Lipids

Abstract

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.

Impact of Embedded Endocannabinoids and Their Oxygenation by Lipoxygenase on Membrane Properties

“…51,52 Furthermore, here we demonstrate that upon LOX-1 binding, AEA-containing liposomes undergo a partial dehydration of the membrane surface, as documented by the spectral shift of the carbonyl stretching band in FTIR analysis. This finding is in line with previous data on the interaction of human 5-LOX with synthetic vesicles 29 and provides further insights into the role of the surface properties of a lipid bilayer on membrane-interacting enzymes, like phospholipases, that undergo interfacial activation by the surrounding lipids. 48 The present data also suggest that HO-AEAs can be generated by LOX-1-dependent oxygenation of membraneembedded AEA and could diffuse laterally within the lipid bilayer to inhibit FAAH, 39 and/or activate NAPE-PLD, 22 thus sustaining a hyper-tone of AEA and subsequently an enhancement of its signaling.…”

“…41 Previous data have indicated that the interaction of LOXs with model membranes can induce an overall stabilization of both the protein and the membrane. 28,29 Here, to dissect the effects of LOX-1 protein matrix from its enzyme activity on membrane properties, we used DPPC liposomes devoid of LOX-1 substrates, as well as AEA-containing LUVs incubated with the inactive apo-form of the enzyme. We found that in both cases, an enzyme concentration range of 0.1−1.0 μM did not affect the T m values of the membrane bilayer (data not shown).…”

Physiology of Cell Volume Regulation in Vertebrates

“…8 (728), and it has been suggested that a PC selectivity of the 5-LO could account for 5-LO targeting to the nuclear membrane (481). Membrane binding per se may, however, not confer 5-LO activity, and although Ca 2ϩ is required for 5-LO activity, 5-LO binds to, e.g., cationic phospholipids in the absence of Ca 2ϩ (761). The 5-LO is subject to phosphorylation by the MAPKs ERK1/2 and p38 MAPK (598,1087,1089), as well as by tyrosine kinases (540).…”

1-Oleoyl-2-acetylglycerol Stimulates 5-Lipoxygenase Activity via a Putative (Phospho)lipid Binding Site within the N-terminal C2-like Domain

“…Ca 2ϩ has been shown to increase the affinity of 5-LO toward AA (24), to facilitate membrane association and binding to PC vesicles (21, 25), and appears to reduce the requirement of 5-LO for activating lipid hydroperoxides (26). For stimulation by phospholipids, apparently only the zwitterionic PC and a non-physiological cationic phospholipid (but not phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, or diacylglycerol (DAG)) stimulate 5-LO reactions (27,28). It has been proposed that 5-LO catalysis occurs at an interface between lipid and water, and the ratio of AA to phospholipid seems important for its activity (29).…”