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 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.
Pande
Abhay H
AH
Biomolecular Science Center, University of Central Florida, Orlando, Florida 32826, USA.
Moe
David
D
Nemec
Kathleen N
KN
Qin
Shan
S
Tan
Shuhua
S
Tatulian
Suren A
SA
D001094:Arachidonate 5-Lipoxygenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D004075:Diglycerides / Q000737:chemistry; D004789:Enzyme Activation / Q000235:genetics; D005989:Glycerides / Q000737:chemistry; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013107:Sphingolipids / Q000737:chemistry; D055672:Static Electricity; D013499:Surface Properties
D002413:Cations, Divalent; D004075:Diglycerides; D005989:Glycerides; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D000644:Quaternary Ammonium Compounds; D013107:Sphingolipids; C034730:zwittergent 3-12; D002784:Cholesterol; D001094:Arachidonate 5-Lipoxygenase; D002118:Calcium; C028694:1-palmitoyl-2-oleoylphosphatidylcholine