The research in this dissertation describes the allosteric activation and inhibition of 5-LOX human lipoxygenase as well as the substrate preferences for all 3 main LOX isozymes, 5-LOX, 12-LOX, and 15-LOX-1, to generate higher-order lipid mediators. 5-LOX is both a hydroperoxidase of arachidonic acid (AA) and an epoxidase of 5(S)-hydroperoxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5(S)-HpETE) to form leukotrienes from a single polyunsaturated fatty acid (PUFA). This dissertation investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE has a significantly lower rate of substrate capture (Vmax/Km) than AA hydroperoxidation, however, hyperbolic kinetic parameters for ATP activation suggest a larger activation with 5(S)-HpETE. Solvent isotope effect (SIE) results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly due a change in the dependency of the rate-limiting step on hydrogen-bond rearrangement toward substrate rearrangement. The products of 5-LOX, leukotrienes and resolvins, both promote and inhibit inflammation, respectively, and therefore changes in ATP concentration in the cell could have wide implications in their relative concentrations and ultimately the regulation of cellular inflammation.
5-LOX inhibition was also profiled with the discovery of a novel dual inhibitor targeting fungal sterol 14alpha-demethylase (CYP51 or Erg11) and 5-LOX. A phenylenediamine core was translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Docking of ketaminazole confirmed kinetic results showing that the drug binds in the active site presenting the phenylenediamine core for effective reduction of the 5-LOX catalytic iron. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.
Epoxidation and hydroperoxidation mechanisms were also investigated in the other main LOX isozymes, 12-LOX and 15-LOX-1. Only 15-LOX-1 was found to effectively hydroperoxidate some of the oxylipins tested, with the hydroxide/hydroperoxide causing a change in the positioning of the substrate in the active site and changing the enzyme's regiospecificity. In contrast, all three LOX isozymes can epoxidate hydroperoxides and this ability is dependent on their respective hydrogen atom abstraction specificity. ATP activation of 5-LOX is also further profiled and found to be more substrate-selective than previously noted. These epoxidation mechanisms may be implicated in higher-order lipids being formed such as lipoxins, eoxins, and hepoxilins, all unique in their ability to mediate inflammation.