CALCIUM IS NOT REQUIRED FOR 5-LIPOXYGENASE ACTIVITY AT HIGH PHOSPHATIDYL CHOLINE VESICLE CONCENTRATIONS

5-Lipoxygenase (5-LO) catalyzes the formation of 5-hydroperoxy-eicosat etraenoic acid (5 HPETE) and leukotriene A(4) (LTA(4)) from arachidoni c acid. Following a rise in intracellular calcium, 5-LO translocates t o a membrane where it reacts with arachidonic acid via an 18 kD protei n (FLAP). In vitro studies using a vesicle system of phosphatidylcholi ne (PC) and purified 5-LO were conducted under varying concentrations of PC and calcium. At high PC concentrations, 5-LO partitioned onto th e vesicle containing arachidonic acid, resulting in product formation in the absence of calcium. Addition of calcium increased the initial r ate of the reaction with a small increase in product accumulation. Dil ution 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 w ithout calcium. Stimulation of 5-LO activity by dithiothreitol (DTT) w as more pronounced at high PC concentrations than at low PC concentrat ions. 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 calc ium. In the presence of PC and calcium and without substrate, the enzy me was unstable and was rapidly and irreversibly inactivated. In high PC without calcium, the enzyme was much more stable but it was still s ubject to turnover-dependent inactivation. Fluorescence energy-transfe r experiments confirmed the kinetic findings that 5-LO could bind to t he vesicle in the absence of calcium. These results show that in the a bsence of calcium, 5-LO can reversibly bind to the vesicle containing arachidonic acid and produce the same amount of product by a similar m echanism as observed with low PC and calcium. Calcium likely causes a conformational change that increases the affinity of the enzyme for th e vesicle, but it is not strictly required for enzymatic activity and has no effect on the function of the catalytic site.