The N-terminal Domain of 5-Lipoxygenase Binds Calcium and Mediates Calcium Stimulation of Enzyme Activity

Abstract

The leukotrienes are important mediators in asthma as well as in other inflammatory and allergic disorders. 5-Lipoxygenase (5-LO 1 ; arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes two initial steps in the cellular production of leukotrienes. Thus, 5-LO converts arachidonic acid into 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently into the unstable epoxide leukotriene A 4 , which, in turn, is the precursor of the biologically active leukotrienes B 4 , C 4 , D 4 , and E 4 (1). Leukotriene B 4 stimulates adherence of leukocytes to the vessel wall and is a potent chemotactic agent for these cells. The cysteinyl leukotrienes C 4 , D 4 , and E 4 increase vascular permeability and are effective constrictors of bronchial smooth muscle. 5-LO also participates in the formation of lipoxins, another group of arachidonate-derived bioactive lipids that are implicated in inflammatory and vascular events (2).Calcium is a well known 5-LO activator (for reviews, see Refs. 3-5). In brief, stimuli that elevate the intracellular Ca 2ϩ levels were shown to induce cellular 5-LO activity, and several reports have described Ca 2ϩ -induced translocation of 5-LO from the cytosol to cellular membranes. More detailed analyses showed an association primarily with the nuclear envelope, where the membrane-bound 5-LO-activating protein (FLAP) is also found and where the substrate arachidonic acid can be released from membrane lipids by cytosolic phospholipase A 2 (cPLA 2 ). The stimulatory effect of Ca 2ϩ is evident also for purified 5-LO. The basal enzyme activity, which is observed in the presence of a membrane fraction or lipids, increases up to 10-fold if micromolar concentrations of Ca 2ϩ are included in the assay mixture. 5-LO catalysis has been shown to occur at the lipid/water interface, and Ca 2ϩ -dependent binding of 5-LO to phospholipid vesicles has been reported. By several experimental approaches, we have recently demonstrated that 5-LO binds Ca 2ϩ in a reversible manner (3). A K d close to 6 M was determined by equilibrium dialysis, and the stoichiometry of maximum binding averaged around two Ca 2ϩ ions/5-LO molecule. We also showed that binding of calcium increased the hydrophobicity of 5-LO. Thus, a present conception is that calcium stimulates 5-LO activity and leukotriene production by promoting membrane association.The first structural determination of a mammalian 15-lipoxygenase (6) revealed that, similar to soybean lipoxygenases (7-9), it is composed of two major domains: a C-terminal domain containing the catalytic site and an N-terminal ␤-barrel domain. It seems reasonable that this is the overall structure also for 5-LO. The capability of 5-LO to bind more than one calcium ion and the calcium-dependent binding to phospholipids make 5-LO functionally similar to a group of calciumbinding proteins known as C2 domain proteins. The C2 domain is a conserved structural motif that forms an eight-stranded anti-parallel ␤-sandwich, and C2 domains have been identified in some 70 membrane-...

Investigations into Calcium-dependent Membrane Association of 15-Lipoxygenase-1

“…Because most LOXs sequenced so far contain an NH 2 -terminal C2-like domain it has been speculated that this structural element may function as phospholipid-binding domain anchoring the enzyme in the lipid bilayer in a calcium-dependent manner (20,21). It should, however, be stressed that the structure of the ␤-barrel domains in LOXs do not exactly match the topology of the C2-domains in phospholipases (20) and thus, there might be differences in the functionality of this structural element.…”

“…SLO1 (12) and the human 5-LOX (20) were suggested to contain such high affinity binding sites but mammalian 15-LOXs have not been investigated in this respect. To address this problem we employed a dual strategy.…”

“…The human 5-LOX binds calcium reversibly with a moderate affinity (K d of about 6 M) and stoichiometry calculations suggested the existence of two specific calcium-binding sites (18). Structural modeling of the enzyme on the basis of the x-ray coordinates for the rabbit 15-LOX (19) suggested that the amino acids interconnecting various ␤-sheets of the NH 2 -terminal domain (Asn 43 , Asp 44 , and Glu 46 ) may constitute poten-tial calcium ligands (20). In fact, mutagenesis of these residues led to an impaired calcium affinity and to requirement for higher calcium concentrations to stimulate enzyme activity (20).…”

“…Structural modeling of the enzyme on the basis of the x-ray coordinates for the rabbit 15-LOX (19) suggested that the amino acids interconnecting various ␤-sheets of the NH 2 -terminal domain (Asn 43 , Asp 44 , and Glu 46 ) may constitute poten-tial calcium ligands (20). In fact, mutagenesis of these residues led to an impaired calcium affinity and to requirement for higher calcium concentrations to stimulate enzyme activity (20). However, as for the soybean enzyme selective calcium binding may not be the only process involved in membrane association.…”

Inhibition of 5-Lipoxygenase Product Synthesis by Natural Compounds of Plant Origin

“…The C2-like domain has a regulatory function, visualized by its ability to bind various lipids [phosphatidylcholine (PC), glycerides or lipid membranes] [29], [30], Ca 2+ or Mg 2+ ions [25], and coactosin-like protein (CLP) [31]. These interactions stimulate 5-LO catalysis in cell-free assays.…”