Non‐Competitive Inhibition by a Substrate Mimetic of Human 5‐Lipoxygenase

Abstract

Human 5‐lipoxygenase (5‐LOX) initiates the synthesis of the leukotrienes (LT) through a two‐step reaction: addition of molecular oxygen to arachidonic acid (AA) into 5–hydroperoxyeicosatetraenoic acid (5‐HPETE) and hydrolysis of 5‐HPETE into leukotriene A4. LTs are highly potent, pro‐inflammatory mediators, which have been implicated in asthmatic and allergic responses. 5‐LOX is mobilized in leukocytes by Ca2+ binding and targets to the membrane to receive substrate from its helper protein, 5‐lipoxygenase‐activating protein (FLAP). These allosteric partners are unnecessary in in vitro experiments but enhance activity in intact cells. The crystal structure of 5‐LOX was revealed in a “closed conformation” with the catalytic iron inaccessible to surface solvent. This closed active site is the result of a unique conformation of helix alpha 2 (Hα2) where two aromatic residues plug the putative substrate‐access portal. 5‐LOX has a disjointed Hα2 of 3 turns connected with multiple loop‐to‐helix transitions permitting this plugged conformation of the active site. For comparison, the structure of another human isoform, 15‐LOX‐2, has Hα2 in a fully elongated conformation of 6 turns and the catalytic iron solvent accessible for substrate entry. The detergent C8E4 was co‐crystallized with 15‐LOX‐2 in the active site, kinetically identified as a competitive inhibitor, and is an excellent substrate mimetic of AA. Initial kinetic studies of 5‐LOX with C8E4 have shown non‐competitive inhibition patterns. We detailed a non‐Michaelis‐Menton kinetic response of 5‐LOX to increasing amounts of substrate at the last EB meeting (https://doi.org/10.1096/fasebj.2020.34.s1.01876). We hypothesized that a ligand‐induced slow transition of the conformation of Hα2 causes the sigmoidal response to substrate by the monomeric enzyme. Kinetic cooperativity of monomeric enzymes with single substrate‐binding sites, while rare, is represented in highly regulated enzymes. We wanted to further elaborate on this model by performing inhibition kinetics of a substrate mimetic of AA to 5‐LOX. Non‐competitive inhibition of an active site binder is further evidence of a slow conformational change, as it indicates a two‐step inhibition mechanism with the slow transition to an inactive enzyme state. Understanding the inhibition kinetics of an enzyme that adopts multiple conformations and detailing the molecular determinants responsible for inhibitor binding could help delineate some of the discrepancies observed for different 5‐LOX inhibitor potencies in different cellular environments. Anti‐leukotriene therapies has proven to be a successful avenue for lowering inflammation in many disease states and more informed structure‐based drug design approach could hasten their development.