Leukotriene A4 hydrolase, mutation of tyrosine 378 allows conversion of leukotriene A4 into an isomer of leukotriene B4.

Leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory compound leukotriene B4, a reaction which is accompanied by suicide inactivation of the enzyme by leukotriene A4. We have recently reported that Tyr-378 is a major structural determinant for suicide inactivation and that mutation of Tyr-378 into Phe or Gln protects leukotriene A4 hydrolase from this catalytic restriction (Mueller, M. J., Blomster, M., Opperman, U. C. T., Jörnvall, H., Samuelsson, B., and Haeggström, J. Z. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5931-5935). In the present study, we show that both [Y378F]- and [Y378Q]leukotriene A4 hydrolase converts leukotriene A4 not only into leukotriene B4 but also into a second, previously unknown, product of the enzyme. From biophysical analyses and comparison with a synthetic standard, the structure of this product was determined to 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e. Δ6-trans-Δ8-cis-leukotriene B4. The relative formation of Δ6-trans-Δ8-cis-leukotriene B4 versus leukotriene B4 by [Y378F]- and [Y378Q]leukotriene A4 hydrolase, was 18% and 32%, respectively. For [Y378F]leukotriene A4 hydrolase, the turnover of leukotriene A4 into leukotriene B4 or Δ6-trans-Δ8-cis-leukotriene B4 was calculated to 2.5 s−1 which is almost three times the kcat value of the wild type enzyme. Taken together, these findings indicate that Tyr-378 is located at the active site where it assists in the formation of the correct double-bond geometry in the product leukotriene B4.

zinc metalloenzyme which converts LTA 4 into the proinflammatory substance LTB 4 , a reaction referred to as the epoxide hydrolase activity (1). In addition, LTA 4 hydrolase possesses an anion-dependent aminopeptidase activity (2)(3)(4), the physiological significance of which is still unknown.
During the epoxide hydrolysis, LTA 4 hydrolase is covalently modified and inactivated by its endogenous lipid substrate LTA 4 via an apparently mechanism-based process, also referred to as suicide inactivation (5)(6)(7)(8). Notably, suicide inactivation is accompanied by loss of both the epoxide hydrolase and the aminopeptidase activity, in agreement with the notion that the corresponding active site(s) share important structural and/or functional elements.
We have recently identified a peptide segment encompassing residues 365-385 in LTA 4 hydrolase to which LTA 4 binds during suicide inactivation (9). A tyrosine residue in position 378 within this peptide appeared to be a primary site for the covalent binding of the lipid to the protein. This conclusion was further corroborated by mutational analysis which revealed that exchange of Tyr-378 for a Phe or Gln rendered the enzyme virtually resistant to mechanism-based inactivation (10). When the mutated enzymes [Y378F]-and [Y378Q]LTA 4 hydrolase were studied in greater detail, it became apparent that they were able to catalyze hydrolysis of LTA 4 not only into the expected product LTB 4 , but also into a novel, previously unknown, enzyme metabolite, as described in the present work. This finding indicates that Tyr-378 is an active site residue, and, in addition, the structure of the novel product gives a clue as to the function of Tyr-378 in the enzymatic conversion of LTA 4 into LTB 4 . 4 6 -tagged fusion proteins in Escherichia coli (JM101) cells, and purified by affinity chromatography on a nickel-nitrilotriacetic acid resin followed by chromatography on hydroxyapatite, as described previously (10).

Materials-LTA
Enzyme Assays-The epoxide hydrolase activity was determined by incubations of enzyme with LTA 4 followed by reverse-phase high performance liquid chromatography (HPLC) analysis of products, as described (11). Quantitations were based on peak area measurements, normalized with respect to the internal standard prostaglandin B 1 (PGB 1 ), using Baseline 810 computer software. The aminopeptidase activity was assayed spectrophotometrically in 50 mM Tris-Cl, pH 7.5, containing 100 mM NaCl and 38 g/ml bovine serum albumin, using 1 mM alanine-4-nitroanilide as the substrate (11).
Gas Chromatography-Mass Spectrometry-Gas chromatography linked to mass spectrometry (GC/MS) was performed with a Hewlett-Packard, model 5970B mass selective detector, connected to a Hewlett-Packard, model 5890 gas chromatograph equipped with a methyl silicone capillary column (length 12 m, film thickness 0.33 m). Helium at a flow rate of 36 cm/s was used as a carrier gas. Injections were made in the split mode at an injector temperature of 200°C. The initial * This work was supported by funds from Swedish Medical Research Council Grants 03X-10350 and 03X-217, European Union Grant BMH4-CT96-0229, Vårdalstiftelsen, and Konung Gustav V:s 80-årsfond. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ Recipient of a fellowship and financial support from the Deutsche Forschungsgemeinschaft.
column temperature was 120°C and was raised at 10°C/min until 240°C. For GC/MS, samples were converted to the methyl ester, trimethylsilyl ether derivatives, by treatment with diazomethane in diethyl ether followed by a mixture of hexamethyldisilazane and trimethylchlorosilane in pyridine.

RESULTS AND DISCUSSION
Previous studies have documented that LTA 4 hydrolase undergoes suicide inactivation during catalysis in a manner indicating a mechanism-based process (7,8). We have recently identified a 21-residue peptide segment (denoted peptide K21) within LTA 4 hydrolase to which LTA 4 binds during suicide inactivation (9). Furthermore, amino acid sequence analysis of a covalently modified form of K21, isolated from LTA 4 hydrolase inactivated by LTA 4 ethyl ester, indicated that Tyr-378 is a primary site for covalent binding of lipid to the protein. To detail the role of Tyr-378 in mechanism-based inactivation of LTA 4 hydrolase, this residue, together with two adjacent serine residues, were subjected to mutational analysis.  (Fig. 1). The UV spectrum of compound IV in MeOH showed a conjugated triene pattern with max at 259, 268, and 279 nm (Fig. 2), i.e. approximately 2 nm hypsochromic to the spectrum of LTB 4 , suggesting that the mutants had produced an isomer of LTB 4 4 hydrolase gave rise to significant amounts of this product (Fig. 1).   (Fig. 2). In GC, compound IV was unstable and eluted as a tailing peak particularly in recordings of the selected ion current of m/z ϭ 213, as previously reported for 5S,12S-DiHETE (13). This GC behavior and the appearance of ion m/z ϭ 213 at high relative intensity, is due to a thermal rearrangement which is typical for compounds with a trans-cis-trans conjugated triene structure (13).

Structure of Compound IV-Compound
To establish the stereochemistry of the hydroxyl groups of compound IV, conjugated cis-double bonds were isomerized, by a short time (Ͻ90 s) exposure to UV light at 310 nm, to produce one of the all-trans isomers of LTB 4 . As shown in Fig. 2, the unknown compound IV was isomerized predominantly into 5S,12R-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic acid (⌬ 6 -trans-LTB 4 ), which was also obtained after isomerization of LTB 4 (data not shown). As a control, the ⌬ 8 -cis double bond of 5S,12S-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid (5S,12S-DiHETE) was isomerized which generated the expected product 5S,12S-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic acid (⌬ 6 -trans-12-epi-LTB 4 ). Thus, the isomerization experiment of compound IV showed that the stereochemistry of the hydroxyl groups must be the same as in LTB 4 , i.e. 5S,12R, and that compound IV has at least one cis double bond probably at ⌬ 8 or ⌬ 10 , since it is different from LTB 4 and its all-trans isomers (compounds I-III, Fig. 1). In principal, a number of possible configurations have to be considered for the doublebond geometry of the conjugated triene of compound IV. However, considering the GC/MS data which indicated the presence of a trans-cis-trans conjugated triene (see above), a single cis double bond at ⌬ 8 seems to be the most likely alternative. The UV spectrum of compound IV ( max at 259, 268, and 279 nm) lended further support for this conclusion. Thus, previous studies have shown that the UV-spectra of LTB 4 and ⌬ 6 -trans-⌬ 10cis-LTB 4 are essentially identical with max ϭ 270 nm whereas isomers carrying a ⌬ 6 -trans-⌬ 8 -cis-⌬ 10 -trans conjugated triene system exhibit spectra with max ϭ 268 nm (14 -16).
A compound with this structure was recently identified in organ homogenates of the African claw toad Xenopus laevis, incubated with LTA 4 (17). In view of the results of the present investigation, it is tempting to speculate that the compound perhaps originated from the action of an isoform of LTA 4 hydrolase present in Xenopus tissues.
Catalytic Properties of Mutated Enzymes-The specific epoxide hydrolase (including formation of compound IV) and peptidase activities of [Y378F]-and [Y378Q]LTA 4 hydrolase are shown in Table I. Both mutants displayed significant activities, although the peptidase activity of [Y378Q]LTA 4 hydrolase was only 1.6% of the wild type enzyme, suggesting that the phenyl moiety of Tyr-378 may play a role in peptidolysis. Nevertheless, it is clear that Tyr-378 is not critical for catalyses, and, in fact, [Y378F]LTA 4 hydrolase exhibited an increased epoxide hydrolase activity (203% of the wild type enzyme, taking the formation of compound IV into account) which might, in part, be due to the absence of the catalytic restriction normally imposed by suicide inactivation. The relative formation of ⌬ 6 -trans-⌬ 8 -cis-LTB 4 versus LTB 4 produced by [Y378F]-and [Y378Q]LTA 4 hydrolase was 17.8 Ϯ 1.1% (mean Ϯ S.D., n ϭ 15) and 32.1 Ϯ 1.3% (mean Ϯ S.D., n ϭ 15), respectively, using an extinction coefficient of 40,000 M Ϫ1 ⅐cm Ϫ1 at 270 nm for ⌬ 6 -trans-⌬ 8 -cis-LTB 4 (cf. Fig. 3). These ratios remained essentially constant under all experimental conditions employed. The mutant [Y378F]LTA 4 hydrolase was subjected to kinetic analysis and was found to have a slightly reduced affinity for LTA 4 (K m ϭ 23.1 M) as compared to wild type enzyme (K m ϭ 5.8 M). On the other hand, the mutant exhibited a higher turnover of LTA 4 into either LTB 4 or ⌬ 6 -trans-⌬ 8 -cis-LTB 4 (k cat ϭ 2.46 s Ϫ1 ) than did the wild type enzyme (0.85 s Ϫ1 ; Table II), and, consequently, the values for k cat /K m were similar. The mutation also increased the K m for the substrate alanine-4-nitroanilide but had essentially no influence on the turnover of this substrate   (15-s) incubations of the respective enzyme (2 g) dissolved in 200 l of 50 mM Tris-Cl, pH 8, with 13 M LTA 4 at room temperature. The products LTB 4 and ⌬ 6 -trans-⌬ 8 -cis-LTB 4 were quantitated with reverse-phase HPLC. The specific peptidase activity was determined from incubations of 2 g of the respective enzyme with 1 mM alanine-4-nitroanilide in 50 mM Tris-Cl, pH 7.5, containing 100 mM NaCl and 38 g/ml bovine serum albumin. Formation of 4-nitroaniline was determined by spectrophotometric analysis at 405 nm.  (Table II). 4 -Leukotriene A 4 is a highly unstable allylic epoxide which is spontaneously hydrolyzed in water with a t1 ⁄2 Ϸ 10 s at neutral pH (18). Nonenzymatic hydrolysis of LTA 4 is thought to be initiated via an acid-induced opening of the epoxide moiety (19) and a carbonium ion, with a positive charge delocalized over the triene system, would be formed as an intermediate in the reaction. This intermediate will result in a planar sp 2hybridized configuration at C12, which in turn allows a nucleophilic attack from both sides of the carbon. Accordingly, the two epimers at C12 of 5S,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic acid, also referred to as ⌬ 6 -trans-LTB 4 and ⌬ 6 -trans-12-epi-LTB 4 will be formed and are in fact the predominant nonenzymatic hydrolysis products of LTA 4 . The structure of LTB 4 , formed by enzymatic hydrolysis, differs from the structure of either of the two nonenzymatically formed 5,12-dihydroxy acids in two ways, viz. the double-bond geometry and the configuration of the hydroxyl group at C12. Apparently, during enzymatic hydrolysis of LTA 4 into LTB 4 , LTA 4 hydrolase ensures a stereoselective introduction of H 2 O at C12 as well as the formation of the thermodynamically less favored ⌬ 6 -cis-⌬ 8trans-⌬ 10 -trans configuration of the conjugated triene. Interestingly, the mutants at position 378 differ from wild type enzyme regarding one of these two essential functions of the enzyme, i.e. the positioning of the cis double bond in the product. Hence, Tyr-378 appears to be involved in this aspect of catalysis, perhaps by assisting in the proper alignment of LTA 4 in the substrate-binding pocket or by promoting a favorable conformation of a putative carbonium ion intermediate. Moreover, the present data, together with our previous findings regarding the role of Tyr-378 in suicide inactivation, strongly indicates that Tyr-378 is an active-site residue.