Characterization of an acetyl‐11‐keto‐β‐boswellic acid and arachidonate‐binding regulatory site of 5‐lipoxygenase using photoaffinity labeling

• Published in: European journal of biochemistry Vol. 256; no. 2; pp. 364 - 368
• Main Authors: Sailer, Eckart‐Roderich, Schweizer, Stephan, Boden, Sven E., Ammon, Hermann P. T., Safayhi, Hasan
• Format: Journal Article
• Language: English
• Published: Berlin & Heidelberg Springer‐Verlag 01.09.1998
• Subjects: 5‐lipoxygenase; Affinity Labels - chemistry; Arachidonate 5-Lipoxygenase - chemistry; arachidonic acid; Arachidonic Acid - metabolism; Azo Compounds - chemistry; Binding Sites - physiology; Binding, Competitive; boswellic acid; Bridged Bicyclo Compounds - metabolism; Calcium - pharmacology; Humans; Leukocytes - enzymology; leukotriene synthesis inhibitor; Lipoxygenase Inhibitors - pharmacology; Molecular Structure; Oleanolic Acid - analogs & derivatives; Quinolines - metabolism; terpenoid; Triterpenes - pharmacology
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.1432-1327.1998.2560364.x

AKBA (acetyl‐11‐keto‐β‐boswellic acid), a natural pentacyclic triterpene, is an orally active leukotriene‐synthesis inhibitor, which acts by a 5‐lipoxygenase−directed, non‐redox, non‐competitive mechanism. It is the only leukotriene‐synthesis inhibitor so far identified that inhibits 5‐lipoxygenase activity as an allosteric regulator and not by a reducing or competitive mechanism. To characterize AKBA's effector site we prepared azido125I‐KBA (4‐azido‐5‐125iodo‐salicyloyl‐β‐alanyl‐11‐keto‐β‐boswellic acid) as a photoaffinity analogue, which inhibited 5‐lipoxygenase activity as efficiently as the lead compound and specifically labeled human 5‐lipoxygenase protein. The labeling of 5‐lipoxygenase by azido‐125I‐KBA strictly depended on the presence of calcium ([Ca2+]free > 500 nM) and was abolished by heat denaturation or by priorincubation with a series of pentacyclic triterpenes (e.g., amyrin, β‐boswellic acid, AKBA and 18α‐glycyrrhetinic acid). In contrast, 18‐β‐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 photoincorporation (IC50 about 10 μM), whereas a variety of other long‐chain fatty acids and their derivatives (e.g., arachidinic acid, arachidonic acid methyl ester, lipoxins A4 and B4) had no effect. The inhibitory arachidonate 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‐lipoxygenase. The AKBA‐binding site is likely to be identical with a regulatory, second arachidonate binding site of the enzyme.