Calcium is not required for 5-lipoxygenase activity at high phosphatidyl choline vesicle concentrations

5-Lipoxygenase (5-LO) catalyzes the formation of 5-hydroperoxy-eicosatetraenoic acid (5 HPETE) and leukotriene A(4) (LTA(4)) from arachidonic acid. Following a rise in intracellular calcium, 5-LO translocates to a membrane where it reacts with arachidonic acid via an 18 kD protein (FLAP). In vitro studies using a vesicle system of phosphatidylcholine (PC) and purified 5-LO were conducted under varying concentrations of PC and calcium. At high PC concentrations, 5-LO partitioned onto the vesicle containing arachidonic acid, resulting in product formation in the absence of calcium. Addition of calcium increased the initial rate of the reaction with a small increase in product accumulation. Dilution experiments in the absence of calcium at high PC concentrations indicated that binding of 5-LO to the vesicles is rapidly reversible. In the presence of calcium, this binding is much more favorable than without calcium. Stimulation of 5-LO activity by dithiothreitol (DTT) was more pronounced at high PC concentrations than at low PC concentrations. The requirement for ATP for maximal activity was independent of vesicle concentration. Inhibitors that functioned in the conditions of low PC with calcium present also inhibited under high PC without calcium. In the presence of PC and calcium and without substrate, the enzyme was unstable and was rapidly and irreversibly inactivated. In high PC without calcium, the enzyme was much more stable but it was still subject to turnover-dependent inactivation. Fluorescence energy-transfer experiments confirmed the kinetic findings that 5-LO could bind to the vesicle in the absence of calcium. These results show that in the absence of calcium, 5-LO can reversibly bind to the vesicle containing arachidonic acid and produce the same amount of product by a similar mechanism as observed with low PC and calcium. Calcium likely causes a conformational change that increases the affinity of the enzyme for the vesicle, but it is not strictly required for enzymatic activity and has no effect on the function of the catalytic site.