Leukotriene A4 (LTA4) hydrolase [(7E,9E, 11Z,14Z)-(5S,6S)-5, 6-epoxyicosa-7,9,11,14- Leukotriene A4 (LTA4) hydrolase (EC 3.3.2.6.) is a bifunctional zinc metalloenzyme which converts LTA4 into the proinflammatory substance LTB4, a reaction referred to as the epoxide hydrolase activity (1). In addition, the enzyme possesses an aminopeptidase activity whose endogenous substrate and physiological significance are unknown (2-4).Previous studies with site-directed mutagenesis have identified His-295, His-299, and Glu-318 as the three zinc-binding ligands (5). With the same technique, Glu-296 was shown to be a catalytic amino acid for the peptidase activity, a finding which allowed us to propose a general base mechanism for the peptide hydrolysis (6). Since the epoxide hydrolase activity was not affected by mutation of Glu-296, the data also showed that the active site(s), corresponding to the two enzyme activities, are not identical although they share several structural and functional properties. Furthermore During catalysis, LTA4 hydrolase is covalently modified and inactivated by its natural lipid substrate LTA4, a process commonly referred to as suicide inactivation (9, 10). LTA4 hydrolase has been proposed to be the rate-limiting enzyme in cellular LTB4 biosynthesis and suicide inactivation may therefore be an important mechanism for the overall regulation of this biosynthetic pathway in vivo (11). Suicide inactivation of LTA4 hydrolase satisfies several criteria of a mechanism-based process ( Fig. 1)

Leukotriene A4 (LTA4) hydrolase [(7E,9E,l1Z, 14Z)-(5S,6S)-5,6-epoxyicosa-7,9,11,14-tetraenoate hydrolase; EC 3.3.2.6] is a bifunctional zinc metalloenzyme which converts LTA4 into the chemotactic agent leukotriene B4 (LTB4).Suicide inactivation, a typical feature of LTA4 hydrolase/aminopeptidase, occurs via an irreversible, apparently mechanism-based, covalent binding of LTA4 to the protein in a 1:1 stoichiometry. Differential lysine-specific peptide mapping of unmodified and suicide-inactivated LTA4 hydrolase has been used to identify a henicosapeptide, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4, LTA4 methyl ester, and LTA4 ethyl ester to the native enzyme. A modified form of this peptide, generated by lysine-specific digestion of LTA4 hydrolase inactivated by LTA4 ethyl ester, could be isolated for complete Edman degradation. The sequence analysis revealed a gap at position 14, which shows that binding of the leukotriene epoxide had occurred via Tyr-378 in LTA4 hydrolase. Inactivation of the epoxide hydrolase and the aminopeptidase activity was accompanied by a proportionate modification of the peptide. Furthermore, both enzyme inactivation and peptide modification could be prevented by preincubation of LTA4 hydrolase with the competitive inhibitor bestatin, which demonstrates that the henicosapeptide contains functional elements of the active site(s). It may now be possible to clarify the molecular mechanisms underlying suicide inactivation and epoxide hydrolysis by site-directed mutagenesis combined with structural analysis of the lipid molecule, covalently bound to the peptide. Leukotriene (LT) B4 is a product of activated leukocytes and has potent autocrine, chemotactic, and chemokinetic properties (1). The final step in the biosynthesis of LTB4 is catalyzed by the enzyme LTA4 hydrolase [(7E,9E,11Z,14Z)-(5S,6S)-5,6-epoxyicosa-7,9,11,14-tetraenoate hydrolase; EC 3.3.2.6), which is a 69-kDa soluble zinc metalloprotein with no apparent structural similarity to other epoxide hydrolases (2). In addition to its well-known epoxide hydrolase activity-i.e., the conversion of LTA4 into LTB4, LTA4 hydrolase possesses an aminopeptidase activity toward synthetic chromogenic amides, opioid peptides, and arginyl tripeptides (3-6), but the endogenous peptidase substrate is still not known. Both activities are dependent on the intrinsic zinc atom and are competitively inhibited by bestatin (7) and captopril (8), classic inhibitors of aminopeptidases and angiotensin converting enzyme, respectively.Previous work has identified several important elements of the active site(s) corresponding to the two enzyme activities. For instance, the intrinsic zinc atom has been shown to be catalytic and complexed to Anions selectively stimulate the peptidase activity, apparently via an anion binding site, and site-directed mutagenesis studies have identified Glu-296 as a catalytic residue which presumably acts as a general base in the peptidase reaction (9). Mu...

Leukotriene A, (LTAJ hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B, and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA, hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site-directed mutagenesis. The mutated cDNAs were expressed in Escherichiu coli and the resulting recombinant proteins, named [Y383F]LTA, hydrolase, [Y383H]LTA, hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA, into leukotriene B,, and the peptidase activity.None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3 % that of the wild-type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LT& hydrolase, [Y383H]LTA, hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10-fold increase in K,,, for leukotriene A, compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with V,,, values of 600 nmol . mg-' . min-' and 1000 nmol . mg-I . min-' for [Y383Q]LTA4 hydrolase and the wild-type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA, hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA, into LTB,.

Leukotriene A, (LTAJ hydrolase is a bifunctional zinc metalloenzyme which catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B, and which also possesses a peptidase activity. From sequence comparisons with aminopeptidases, a tyrosine at position 383 in LTA, hydrolase has been suggested as a possible catalytic amino acid. To explore the potential role of this amino acid in catalysis, we replaced the tyrosine residue with phenylalanine, histidine or glutamine residues by site-directed mutagenesis. The mutated cDNAs were expressed in Escherichiu coli and the resulting recombinant proteins, named [Y383F]LTA, hydrolase, [Y383H]LTA, hydrolase and [Y383Q]LTA4 hydrolase, were purified to homogeneity to allow assays of both the epoxide hydrolase activity, i.e. the conversion of LTA, into leukotriene B,, and the peptidase activity.None of the mutated proteins exhibited significant peptidase activities, all of them showing activities less than 0.3 % that of the wild-type enzyme. The epoxide hydrolase activity was not affected to the same degree and corresponded to 11, 16 and 17% that of the unmutated enzyme for [Y383F]LT& hydrolase, [Y383H]LTA, hydrolase and [Y383Q]LTA4 hydrolase, respectively. Kinetic analysis was performed with the mutant [Y383Q]LTA4 hydrolase, which revealed an approximately 10-fold increase in K,,, for leukotriene A, compared to that for the unmutated enzyme. At high concentrations of substrate, the difference in enzyme velocity was only moderate, with V,,, values of 600 nmol . mg-' . min-' and 1000 nmol . mg-I . min-' for [Y383Q]LTA4 hydrolase and the wild-type enzyme, respectively. No such effect of substrate concentration could be observed on the peptidase activity. As a positive control, we exchanged a glycine residue in position 386 for an alanine residue, and the recombinant protein, [G386A]LTA4 hydrolase retained 19% and 77% of the peptidase and epoxide hydrolase activities, respectively. The results from this study are consistent with a role for Tyr383 in the peptidase reaction of LTA, hydrolase, where it may act as a proton donor in a general base mechanism. However, our data do not allow a similar interpretation for the mechanism involved in the hydrolysis of LTA, into LTB,.

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