CHARACTERIZATION OF AN ACETYL-11-KETO-BETA-BOSWELLIC ACID AND ARACHIDONATE-BINDING REGULATORY SITE OF 5-LIPOXYGENASE USING PHOTOAFFINITY-LABELING

AKBA (acetyl-11-keto-beta-boswellic acid), a natural pentacyclic trite rpene. is an orally active leukotriene-synthesis inhibitor, which acts by a 5-lipoxygenase-directed, non-redox, non-competitive mechanism. I t is the only leukotriene-synthesis inhibitor so far identified that i nhibits 5-lipoxygenase activity as an allosteric regulator and not by a reducing or competitive mechanism. To characterize AKBA's effector s ite we prepared azido(125)I-KBA iodo-salicyloyl-beta-alanyl-11-keto-be ta-boswellic acid) as a photoaffinity analogue, which inhibited 5-lipo xygenase activity as efficiently as the lead compound and specifically labeled human 5-lipoxygenase protein. The labeling of 5-lipoxygenase by azido-I-125-KBA strictly depended on the presence of calcium ([Ca2](free) > 500 nM) and was abolished by heat denaturation or by priorin cubation with a series of pentacyclic triterpenes (e.g., amyrin, beta- boswellic acid, AKBA and Isa-glycyrrhetinic acid). In contrast, 18-bet a-glycyrrhetinic acid and competitive 5-lipoxygenase inhibitors (e.g., ZM-230,487 and L-739,010) did not affect labeling. Arachidonic acid, in enzyme-activity-inhibiting concentrations, reduced photoincorporati on (IC50 about 10 mu M). whereas a variety of other long-chain fatty a cids and their derivatives (e.g., arachidinic acid, arachidonic acid m ethyl ester. lipoxins A(4) and B-4) had no effect. The inhibitory arac hidonate action on labeling was not affected by blocking the substrate -binding site by micromolar amounts of the competitive inhibitor L-739 ,010. Therefore, we suggest that AKBA binds in presence of calcium to a site which is distinct from the substrate binding site of 5-lipoxyge nase. The AKBA-binding site is likely to be identical with a regulator y, second arachidonate binding site of the enzyme.