Modulation of Human 5-Lipoxygenase Activity by Membrane Lipids

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca2+-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of mem...

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Veröffentlicht in:Biochemistry (Easton) 2004-11, Vol.43 (46), p.14653-14666
Hauptverfasser: Pande, Abhay H, Moe, David, Nemec, Kathleen N, Qin, Shan, Tan, Shuhua, Tatulian, Suren A
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Sprache:eng
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Zusammenfassung:Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca2+-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 Ca2+, there was substrate inhibition at >100 μM AA. Data analysis at low AA concentrations yielded the following:  K m ≈ 103 μM and k cat ≈ 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 Ca2+, whereas binding to cationic membranes was stronger and essentially Ca2+-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 β-barrel tilted ∼45° 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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi048775y