Leukotriene (LT) A4 hydrolase/aminopeptidase is a bifunctional zinc enzyme that catalyzes the final step in the biosynthesis of LTB4, a potent chemoattractant and immune modulating lipid mediator. Here, we report a high-resolution crystal structure of LTA4 hydrolase in complex with captopril, a classical inhibitor of the zinc peptidase angiotensin-converting enzyme. Captopril makes few interactions with the protein, but its free thiol group is bound to the zinc, apparently accounting for most of its inhibitory action on LTA4 hydrolase. In addition, we have determined the structures of LTA4 hydrolase in complex with two selective tight-binding inhibitors, a thioamine and a hydroxamic acid. Their common benzyloxyphenyl tail, designed to mimic the carbon backbone of LTA4, binds into a narrow hydrophobic cavity in the protein. The free hydroxyl group of the hydroxamic acid makes a suboptimal, monodentate complex with the zinc, and strategies for improved inhibitor design can be deduced from the structure. Taken together, the three crystal structures provide the molecular basis for the divergent pharmacological profiles of LTA4 hydrolase inhibitors. Moreover, they help define the binding pocket for the fatty acid-derived epoxide LTA4 as well as the subsites for a tripeptide substrate, which in turn have important implications for the molecular mechanisms of enzyme catalyses.

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A methionine-containing tryptic peptide from acetamidinated human carbonic anhydrase B has been isolated. This peptide has been shown to bridge a previously known carboxyl-terminal fragment prepared by CNBr degradation with the rest of the enzyme molecule. Sequence studies on the methionine-containing peptide have extended the previous knowledge of the carboxylterminal sequence of the enzyme.Corresponding bridge peptides from human carbonic anhydrase C and acetamidinated bovine carbonic anhydrase B have also been isolated and their amino acid compositions have been determined.The preceding paper [l] gives the carboxyl-terminal sequences of human carbonic anhydrase forms B and C and bovine carbonic anhydrase formB, (for nomenclature see [2]). The results were obtained by characterization of carboxyl-terminal peptides derived from the proteins by splitting with CNBr. From the specificity of this reagent [3] one would expect a methionine residue to be located next to the sequences determined.The present investigation is a study of methioninecontaining tryptic peptides bridging the previously determined carboxyl-terminal sequence with t,he rest of the enzyme molecule. MATERIALS AND METHODS Preparation of Carbonic AnhydrasesHuman carbonic anhydrases B and C, and bovine carbonic anhydrase B were purified as described previously [l]. Acetamidination of Human Carbonic Anhydrase B and Bovine Carbonic Anhydrase BAcetamidination was performed as described elsewhere [4]. Proteolytic Digestions with EndopeptidasesTryptic digestions were performed a t room temperature in a pH-stat (Radiometer, Copenhagen : Titrator, type TTTlc, in combination with Titri- Unusual Abbreviations. Di-isopropyl phosphorofluoridate, DFP; 2,4-dinitrophenyl, Dnp-; sulfoethyl, SE-; glntamine or glutamic acid, Glx. graph, typeSBR2c) a t pH 9.0 with trypsin (3 x crysf., salt free, TRL 61 13, Worthington Biochemical Corporation, Freehold, New Jersey 07728) treated with diphenyl-carbamyl chloride [5]. A 1 solution of human carbonic anhydrase C in water was adjusted to pH 9.0 with 0.05 M NaOH. The protein was denatured by keeping the solution in a boiling water bath for 15 min. Trypsin, dissolved in 2.5 mM HC1, was added. After 6 h another amount of trypsin solution was added. After about 10 h from start, the consumption of NaOH had almost ceased. The heat denaturation was then repeated and an additional aliquot of trypsin was added. The total amount of trypsin added then corresponded to a molar ratio of substrate/trypsin of 100. When no more NaOH was consumed (usually after 13-14 h) pH was decreased to about 3 with glacial acetic acid.Tryptic digestions of acetamidinated human enzyme B and bovine enzyme B were performed as described for human enzyme C but the final molar ratio of substrate/trypsin was 50 and the initial concentration of substrate 1.5-2 Oi0. Under these conditions the hydrolyses took 4.5-6 h.A digestion of a peptide with subtilisin was carried out by dissolving the peptide (9.4 pmoles) in 2.5 ml of 0.1 M Tris-C1 buffer, pH 9.0, and adding...

Die Titelverbindungen werden zwecks Untersuchung der Bedingungen für die spezifische trypsinkatalysierte Hydrolyse der His°"‐Ser°5‐Bindung dargestellt.