Analysis of the Molecular Mechanism of Substrate-mediated Inactivation of Leukotriene A4 Hydrolase*

Martin J. MuellerDagger , Martina Andberg, and Jesper Z. Haeggström§

Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The bifunctional leukotriene A4 hydrolase catalyzes the final step in the biosynthesis of the proinflammatory leukotriene B4. During exposure to the substrate leukotriene A4, a labile allylic epoxide, the enzyme is gradually inactivated as a consequence of the covalent binding of leukotriene A4 to the active site. This phenomenon, commonly referred to as suicide inactivation, has previously been rationalized as a mechanism-based process in which the enzyme converts the substrate to a highly reactive intermediate within an activated enzyme-substrate complex that partitions between covalent bond formation (inactivation) and catalysis. To further explore the molecular mechanism of the self-inactivation of leukotriene A4 hydrolase by leukotriene A4, we prepared and analyzed mutated forms of the enzyme that were either catalytically incompetent or fully active but resistant toward substrate-mediated inactivation. These mutants were treated with leukotriene A4 and leukotriene A4 methyl and ethyl esters and subjected to differential peptide mapping and enzyme activity determinations, which showed that inactivation and/or covalent modification can be completely dissociated from catalysis. Our results, together with recent findings described in the literature, argue against a mechanism-based model for suicide inactivation. We conclude that the collected data on the substrate-mediated inactivation of leukotriene A4 hydrolase best conforms to an affinity-labeling mechanism.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Leukotriene (LT)1-A4 hydrolase (EC. 3.3.2.6.) is a bifunctional zinc metalloenzyme that 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 toward a variety of substrates including synthetic chromogenic amides (2), opioid peptides (3), and arginyl tripeptides (4). However, the endogenous substrate and the physiological significance of the peptidase activity are still not known.

During catalysis, LTA4 hydrolase converts LTA4 stereospecifically to LTB4, but a fraction of the substrate will covalently bind to the active site of the enzyme, causing irreversible inactivation of both enzyme activities (5, 6). This process, usually referred to as suicide inactivation, has been proposed to be rate-limiting in cellular LTB4 biosynthesis and may therefore be an important mechanism for the overall regulation of this biosynthetic pathway in vivo (7). Using differential Lys-specific peptide mapping, we have recently identified a heneicosapeptide, denoted K21, encompassing the amino acid residues 365-385 of human LTA4 hydrolase, which is involved in the binding of LTA4 and LTA4 methyl and ethyl ester to the native enzyme. In experiments using LTA4 ethyl ester, it was possible to identify Tyr-378 as the residue that is involved in the binding of the lipid (8). In a recent study using site-directed mutagenesis, we could show that a conservative replacement at position 378 increased the turnover for epoxide hydrolysis and rendered the enzyme virtually resistant to suicide inactivation with LTA4 (9). Thus, the function of Tyr-378 was not primarily catalytic since (Y378F/Q)LTA4 hydrolase displayed both enzyme activities. However, the hydrolysis of LTA4 catalyzed by the mutated enzymes was affected, leading to a mixture of LTB4 and 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid in a ratio of 1:0.2 for Y378F and 1:0.3 for Y378Q-LTA4 hydrolase, suggesting that the transition state complexes formed with the mutated enzymes are less well defined as compared with that of the wild type enzyme (10).

It has been proposed that the substrate-mediated inactivation of LTA4 hydrolase by LTA4 is a mechanism-based process (Fig. 1A), which would imply that the inactivation (i.e. covalent modification) is dependent on turnover and vice versa (5, 6). In order to test this hypothesis, we investigated the interaction of the catalytically incompetent E318A-LTA4 hydrolase with LTA4 and LTA4 methyl and ethyl ester. In addition, we investigated the mutated enzymes Y378F and Y378Q-LTA4 hydrolase that are resistant toward substrate-mediated inactivation. Our results indicate that LTA4 is an affinity label rather than a mechanism-based inhibitor that modifies and blocks the active site by virtue of its binding specificity and inherent chemical reactivity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- LTA4 methyl ester was from Biomol, and LTA4 ethyl ester was from Merck-Frosst Laboratories, Pointe Claire, Quebec, Canada. Alanine-4-nitroanilide, iodoacetic acid sodium salt, and dithiothreitol were from Sigma. LTA4 ethyl ester was saponified in tetrahydrofuran with 1 M LiOH (6% v/v) for 48 h at 4 °C. T7 sequencing kit, restriction endonucleases, T4 DNA ligase, and PD-10 columns were purchased from Amersham Pharmacia Biotech. Nickel nitrilotriacetic acid resin was from Qiagen. Oligonucleotides were synthesized by Scandinavian gene synthesis. Lys-C protease (150 units/mg, Boehringer Mannheim) was dissolved in 1 mM HCl and stored up to 4 weeks at -20 °C.

Site-directed Mutagenesis of Human LTA4 Hydrolase-- Y378F- and Y378Q-LTA4 hydrolase were constructed by polymerase chain reaction mutagenesis on human LTA4 hydrolase cDNA as described previously (9). The mutated cDNAs were cloned into the Escherichia coli expression vector pT3-MB4, which generates a His6-tagged fusion protein. E318A-LTA4 hydrolase was previously constructed by oligonucleotide-directed mutagenesis on single-stranded DNA, as described (11). A BglII/AflII fragment containing the mutated codon was excised from the cDNA and ligated into the expression vector pT3-MB4, previously opened with the same restriction enzymes. The integrity of the final expression construct was verified by DNA sequencing using the dideoxy chain termination method (12).

Purification of Recombinant LTA4 Hydrolase-- Mutated proteins were expressed in E. coli (JM101) and purified to apparent homogeneity by affinity chromatography on a nickel nitrilotriacetic acid resin followed by hydroxyapatite chromatography, as described (9).

Enzyme Assays-- The epoxide hydrolase activity was determined from incubations of 2 µg of LTA4 hydrolase in 200 µl of 50 mM Tris-Cl, pH 8, with 1 µl of the substrate LTA4 (2.4-2.8 nmol/µl) at room temperature. The reaction was stopped after 15 s by the addition of 2 volumes of methanol, and prostaglandin B1 was added as an internal standard. The samples were acidified to pH 3 with 0.1 M HCl followed by solid phase extraction on Chromabond C18 columns (Waters). The formation of LTB4 was analyzed by reverse phase HPLC, and the quantitations were made from area integration based on a standard curve obtained from analysis of known amounts of prostaglandin B1 and LTB4 (13).

The aminopeptidase activity was determined spectrophotometrically in 50 mM Tris-Cl, pH 7.5, containing 100 mM NaCl and 38 µg/ml bovine serum albumin with 1 mM alanine-4-nitroanilide as substrate. The reactions were performed in the wells of a microtiter plate with 2 µg of purified enzyme, and product formation was measured as an increase in the absorbance at 405 nm, using a multiscan spectrophotometer. Spontaneous hydrolysis of the substrate was corrected for by subtracting the absorbance of control incubations without enzyme (13).

Inactivation Experiments and Peptide Mapping-- Wild type and mutated LTA4 hydrolase (2 ml, 100 µg/ml in 50 mM Tris-Cl, pH 8) was incubated five times with 14 µM LTA4, LTA4 methyl ester, or LTA4 ethyl ester for 30 min at room temperature (final concentration 70 µM). A portion of the reaction mixture (100 µl) was then gel-filtered on a PD-10 column, and the eluted protein was analyzed for residual epoxide hydrolase and peptidase activity. The remaining enzyme was subjected to Lys-C digestion and differential peptide mapping by reverse phase HPLC as described (8). Peptides K21 and Y378F-K21 were identified by amino acid sequence analysis (8, 9). The presence (or loss) of K21 or (Y378F/Q)K21 in samples of enzyme treated with LTA4 or LTA4 methyl or ethyl ester was calculated from the peak height at 280 nm in peptide maps as described (8). Values were normalized with respect to neighboring peaks to compensate for variations in recovery and were expressed in percent of the corresponding peak height obtained with untreated enzyme.

For separate tests of enzyme inactivation, small-scale (100 µl) incubations of LTA4 hydrolase (100 µg/ml in 50 mM Tris-Cl, pH 8) with 3 × 2.6 µM LTA4 (final concentration 78 µM) were performed followed by gel filtration (PD-10) and activity determinations.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Covalent Modification of E318A-LTA4 Hydrolase-- Glutamic acid at position 318 is the third zinc binding ligand in the active site of LTA4 hydrolase (1). Accordingly, E318A-LTA4 hydrolase has been shown to lack the catalytic zinc atom and is therefore virtually devoid of epoxide hydrolase and aminopeptidase activity (11, 14). We utilized this mutant to investigate if a catalytically incompetent enzyme could be modified by the substrate LTA4 or its ester derivatives. To this end, E318A-LTA4 hydrolase (200 µg in 2 ml of Tris-Cl, pH 8, 10 mM EDTA) was exposed to five consecutive additions of 14 µM LTA4 at 30-min intervals and processed as described above. Peptide maps of untreated E318A-LTA4 hydrolase and E318A exposed to LTA4 were identical except for peptide K21, previously identified as the peptide to which LTA4 binds during suicide inactivation (8, 9). Thus, in peptide maps of enzyme treated with LTA4, K21 was reduced to approximately 25% of the untreated control (see Fig. 2; Table I). In addition, novel peaks appeared in the more lipophilic region of the chromatogram, in line with the notion that modification of the active-site peptide K21 had occurred (data not shown). Interestingly, for the catalytically active wild type enzyme, the modification as well as the corresponding inactivation by LTA4 did not exceed 50% of the untreated control (see Fig. 3) (8).

                              
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Table I
Inactivation of wild type and Y378F-LTA4 hydrolase: Residual activities and modification of peptide K21 or Y378F-K21 spanning Leu-365-Lys-385
Enzymes were treated with five consecutive additions of 14 µM leukotriene epoxides. After gel filtration, aliquots of the enzyme were tested for enzyme activities. The remaining enzyme was subjected to Lys-C digestion and differential peptide mapping by reverse phase HPLC.

Under the same conditions, LTA4 methyl or ethyl ester reacted with E318A-LTA4 hydrolase in a similar way, leading to a specific modification of peptide K21 as judged by its disappearance (remaining peptide 18 and 10% of the control, respectively) in the corresponding peptide maps (Table I). Notably, the extent of peptide modification when E318A-LTA4 hydrolase was treated with LTA4 methyl and ethyl ester was comparable to the corresponding modification of peptide K21 using wild type enzyme (23 and 14% of the control, respectively). In all experiments with peptide mapping of E318A-LTA4 hydrolase, the low recovery of modified peptides prevented their isolation and amino acid sequencing. Hence, the site of attachment between lipid and protein could not be determined.

Treatment of (Y378F/Q)LTA4 Hydrolase with LTA4 Methyl and Ethyl Ester-- The three allylic epoxides LTA4, LTA4 methyl ester, and LTA4 ethyl ester are all effective inhibitors of LTA4 hydrolase, but the latter two compounds do not generate detectable amounts of products and are therefore generally not regarded as substrates for LTA4 hydrolase. Nevertheless, based on kinetic data, inactivation of the enzyme by LTA4 methyl ester has been interpreted as a mechanism-based process, albeit with negligible turnover (6). With this interpretation, the rate of inactivation would by far exceed the rate of product formation (cf. Fig. 1A). Since Y378F-LTA4 hydrolase is resistant to suicide inactivation and exhibits an increased turnover of LTA4 (9), this enzyme could potentially be more efficient in converting the ester derivatives of LTA4 into product. However, in our activity assays (1 µg of enzyme in 100 µl, 50 mM Tris-Cl, pH 8, incubated with 2.6 nmol of LTA4 methyl or ethyl ester) Y378F-LTA4 hydrolase failed to generate any detectable amounts of the corresponding esters of LTB4. Moreover, when the amounts of enzyme and LTA4 esters were increased to 20 µg (28 nmol) and 76 nmol, respectively, and the time of incubation was extended to 20 min, no formation of enzymatic products could be detected. Hence, under these seemingly optimized conditions, we could not detect any enzymatic turnover of the ester derivatives of LTA4, suggesting that these compounds are neither substrates nor mechanism-based inhibitors of LTA4 hydrolase.


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Fig. 1.   Schematic representation of mechanism-based inactivation (A) versus affinity-labeling (B). Scheme A depicts mechanism-based inactivation of LTA4 hydrolase. LTA4 forms an enzyme-substrate complex and, within this complex, is enzymatically converted to a highly reactive species, LT*. LT* may either be subject to a second enzymatic transformation to form LTB4 or may covalently bind to an active-site residue. Scheme B exemplifies an affinity-labeling mechanism. The electrophilic LTA4 is noncovalently bound to the active site, from where it captures a proximate residue without any catalytic assistance by the enzyme. This process may be suppressed to a certain extent by the enzymatic conversion of the reactive LTA4 to LTB4. The partition ratio (turnover/inactivation) is determined by the rate constants k3 and k4 (5) in scheme A or the rate constants k2 and k3 in scheme B, respectively.

To investigate if (Y378F/Q)LTA4 hydrolase were resistant also to inactivation with LTA4 methyl and ethyl ester, the effects of treatment with these leukotriene epoxides were compared with the inactivation pattern observed for wild type enzyme (Table II). The two mutants S379A- and S380A-LTA4 hydrolase, which are not protected from suicide inactivation (9), were prepared and treated under the same conditions to serve as controls. Surprisingly, partial inactivation of the epoxide hydrolase activities of Y378F- and Y378Q-LTA4 hydrolase was observed although not to the same extent as for the wild type enzyme and the two control mutants S379A- and S380A-LTA4 hydrolase (Table II). Thus, for Y378F- and Y378Q-LTA4 hydrolase, inactivation of the epoxide hydrolase activity with LTA4 methyl ester was significant, yielding 52 and 44% inhibition, respectively. The corresponding effect of LTA4 ethyl ester was less pronounced, with 40 and 16% inhibition for Y378F and Y378Q, respectively. Under the same incubation conditions, the control enzymes lost about 70 and 80% of the initial epoxide hydrolase activity by treatment with LTA4 methyl and ethyl ester, respectively.

                              
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Table II
Inactivation of wild type and mutated forms of LTA4 hydrolase: Residual epoxide hydrolase and aminopeptidase activity after treatment with LTA4 methyl ester or LTA4 ethyl ester
Enzymes were treated with three consecutive additions of 2.6 µM LTA4 and separated from the enzymatic and nonenzymatic reaction products by gel filtration before the remaining enzyme activities were determined as described under "Experimental Procedures."

Typically, treatment of wild type and S379A- and S380A-LTA4 hydrolase with LTA4 or LTA4 methyl or ethyl ester results in an equal and parallel loss of both enzyme activities (9) (Table II). However, the peptidase activities of Y378F- and Y378Q-LTA4 hydrolase were not inactivated but rather stimulated by treatment with the ester compounds (except for LTA4 ethyl ester, which slightly inhibited the peptidase activity of Y378F-LTA4 hydrolase), indicating that these mutants become modified at a different position within the active site (see below).

Peptide Mapping of Inactivated Enzymes-- To determine the effects of mutation of Tyr-378 on covalent modification of LTA4 hydrolase by LTA4 methyl and ethyl ester, large scale inactivation experiments (200 µg of enzyme) with subsequent proteolytic cleavage and HPLC peptide mapping were performed. The modification of the active-site peptide K21 or Y378F-K21 was determined from the reduction of the corresponding peptide peak in the peptide maps. Although Y378F-LTA4 hydrolase was neither inactivated nor modified by LTA4 (9) (Fig. 3), LTA4 methyl and ethyl ester did inactivate the epoxide hydrolase activity and modify Y378F-LTA4 hydrolase, although not to the same extent as observed for the wild type enzyme (Table I and II; Fig. 3). Furthermore, the inactivation of the epoxide hydrolase activity of Y378F-LTA4 hydrolase appeared to be reflected by a proportional loss of peptide Y378F-K21 (Table I), indicating a modification within the same peptide segment as in the unmutated enzyme but yet at a different position (see above). Novel peaks, which eluted late in the peptide maps, appeared in analyses of enzyme that had been inactivated with ester derivatives of LTA4 (data not shown). These peaks may be lipid-protein adducts that elute late as compared with the corresponding free peptides due to their increased hydrophobicity. However, because of the low recovery, those peptides could not be isolated and sequenced.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

LTA4, the key precursor of the biologically active leukotrienes, is an unusual example of an endogenous compound that is both a natural substrate and an irreversible inactivator of a target enzyme, i.e. LTA4 hydrolase. Results obtained by several experimental approaches have indicated that this inactivation of LTA4 hydrolase satisfies criteria of a mechanism-based process (6) (Fig. 1A). Thus, after treatment of native and recombinant human LTA4 hydrolase with LTA4 or LTA4 methyl ester, the epoxide hydrolase and peptidase activities are lost simultaneously and irreversibly in a time-dependent, saturable process that is of pseudo first-order kinetics. There is a direct relationship between catalysis and inactivation with equivalent pH dependence for both processes. Active-site specificity has been demonstrated by protection with competitive inhibitors (6, 8, 15), and mass spectrometric analysis has revealed that suicide inactivation occurs predominantly in a 1:1 stoichiometry between lipid and protein, with only little modification of secondary sites (6). From these data, substrate-mediated inactivation of LTA4 hydrolase has been classified as a mechanism-based process. However, all these criteria are also consistent with an affinity-labeling mechanism (Fig. 1B).

Mechanism-based Inhibitors Versus Affinity Labels-- A mechanism-based inhibitor is an unreactive molecule that is activated by the catalytic machinery of an enzyme into a reactive product which in turn will covalently modify an active-site residue. Typically, a mechanism-based inhibitor is a synthetically prepared substrate analogue that has been designed to covalently bind a catalytic acid-base of the transition state after its enzymatic transformation into a reactive species (16, 17). Since the mechanism-based inhibitor requires the catalytic apparatus for its action, this type of compound is ideal for the development of highly specific enzyme inhibitors.

In contrast, an affinity label is a molecule that is chemically reactive in itself and therefore does not need enzymatic processing to become destructive. This type of inhibitor, which resembles the substrate, will also modify the active site because of its binding specificity. On the other hand, an affinity label could potentially react with any protein to which it binds with sufficient strength. Thus, the distinction between a mechanism-based inhibitor and an affinity label has important pharmacological and biological implications, and the two modes of inactivation can be schematically illustrated as in Fig. 1.

Apparently, a critical point in deciding which mechanism best describes the substrate-mediated inactivation of LTA4 hydrolase will be to determine whether or not the enzyme machinery is required for generation of the reactive inactivating molecule. One possible species could be a carbocation, which is formed during nonenzymatic hydrolysis of LTA4 and which also seems to be an intermediate in the conversion of LTA4 into LTB4 (18).

The Catalytically Inactive Mutant E318A-LTA4 Hydrolase Is Covalently Modified by LTA4-- Mutagenetic replacement of any of the three zinc binding ligands in LTA4 hydrolase including Glu-318 leads to the combined loss of zinc and enzyme activities (11). Thus, the mutant E318A-LTA4 hydrolase was selected to study the effects of leukotriene epoxides on an enzyme lacking a key element of catalysis, the fundamental basis for a mechanism-based process. Exposure of E318A-LTA4 hydrolase to LTA4 or LTA4 methyl or ethyl ester resulted in covalent modification of peptide K21 to the same extent as for wild type enzyme (Figs. 2 and 3; Table I). This finding is very difficult to rationalize within the concept of a mechanism-based process in which catalytic activation of the substrate is necessary and precedes covalent modification and inactivation. In fact, the degree of modification was even higher in the absence of catalysis (cf. Figs. 2 and 3). Given an affinity-labeling mechanism, the latter observation can be explained by the enzymatic capacity of LTA4 hydrolase to convert the reactive substrate to the stable end product LTB4, thereby suppressing the destructive side reaction with the protein.


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Fig. 2.   Differential peptide mapping of untreated E318A-LTA4 hydrolase and E318A treated with LTA4 or LTA4 ethyl ester. The HPLC chromatograms (35-60 min; 280 nm) were obtained from peptides generated by Lys-specific digestion of untreated E318A-LTA4 hydrolase or enzyme treated with the leukotrienes, as described under "Experimental Procedures." The arrow indicates the position of the remaining unmodified peptide K21.


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Fig. 3.   Differential peptide mapping of untreated wild type (WT) and Y378F-LTA4 hydrolase versus enzymes treated with LTA4 or LTA4 ethyl ester. The HPLC chromatograms (35-60 min; 280 nm) were obtained from peptides generated by Lys-specific digestion of wild type and Y378F-LTA4 hydrolase with or without pretreatment with the leukotrienes, as described under "Experimental Procedures." The arrow indicates the position of the remaining unmodified peptide K21 and Y378F-K21, respectively.

Covalent Modification of a Nonessential Residue at the Active Site of LTA4 Hydrolase-- Tyr-378 has been identified as a major site to which LTA4 binds during suicide inactivation (8). Exchange of Tyr-378 for a Phe or Gln rendered the enzyme virtually resistant to suicide inactivation and, in addition, Y378F-LTA4 hydrolase exhibited an increased kcat for LTA4 (9). Hence, turnover was completely dissociated from inactivation, which shows that Tyr-378 is not critical for catalysis. Modification of a nonessential active-site residue would be consistent with an affinity-labeling mechanism (17) in which LTA4 may simply become captured by a proximate nucleophilic residue at the active site, namely Tyr-378.

Leukotriene Epoxides Bind to More Than One Site within Peptide K21-- Although Y378F- and Y378Q-LTA4 hydrolases were not susceptible to covalent modification/inactivation by LTA4 (9), these two mutants were only partially protected against inactivation by LTA4 methyl and ethyl ester (Table II, Fig. 3), none of which was turned over into a detectable product. Thus, after treatment with esterified LTA4, the epoxide hydrolase activities were reduced, albeit not to the same extent as for wild type enzyme, whereas the peptidase activities were almost unaffected or, in the case of Y378Q-LTA4 hydrolase, even stimulated (Table II). Furthermore, experiments with peptide mapping of Y378F-LTA4 hydrolase treated with the ester derivatives of LTA4 showed that the modification had occurred within peptide K21, which represents at least a part of the substrate binding pocket (Fig. 3). Inasmuch as suicide inactivation of wild type LTA4 hydrolase typically affects both activities to the same extent, the selective inhibition of the epoxide hydrolase activity, without concomitant loss of the peptidase activity, lends further support to the conclusion that the lipid/protein adduct has been formed at a different site in the active center. Hence, although Tyr-378 appears to be best positioned for modification by LTA4, other nucleophilic residues along K21 may also react with the epoxide, at least in the case of esterified LTA4. Moreover, the unproductive binding of LTA4 methyl and ethyl ester to the active site (no detectable formation of LTB4 esters) and the tendency to form several alternative covalent bonds would also be consistent with an affinity-labeling mechanism.

Molecular Mechanism of Substrate-mediated Inactivation of LTA4 Hydrolase-- Based on primarily kinetic data, suicide inactivation of LTA4 hydrolase has been interpreted as a mechanism-based process (5, 6). However, this model is difficult to reconcile with certain properties of LTA4. For instance, the molecule LTA4, its ester derivatives, and structurally related allylic epoxides are all chemically labile compounds that react indiscriminately with all nucleophiles available. In fact, the half-life of LTA4 is less than 10 s in aqueous solutions (25 °C, phosphate buffer, pH 7.4) (19). Hence, an additional step of substrate activation does not seem to be required for covalent bond formation/inactivation. The importance of the chemical reactivity of the allylic epoxide in LTA4 for suicide inactivation is also underscored by the fact that other structurally dissimilar allylic epoxides (20) also inactivate LTA4 hydrolase, whereas non-allylic epoxides do not (Fig. 4).


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Fig. 4.   Structurally related epoxides that can or cannot act as substrates and/or inhibitors of LTA4 hydrolase. LTA3 (a), LTA4 (b), and LTA5 (c) are all substrates as well as inhibitors of LTA4 hydrolase. Other structural analogues of LTA4 that contain an allylic epoxide and that are not accepted as substrates, e.g. 9-cis-11-trans-LTA4 (d), LTA4 methyl ester (e), and 14,15-LTA4 (f) are still efficient inhibitors of LTA4 hydrolase. Epoxides that do not contain an allylic epoxide, e.g. 8,10,14-cis-12-trans-LTA4 (g), 11,12-epoxy-eicosatrienoic acid (h), and 14,15-epoxy-eicosatrienoic acid (i) are neither substrates nor inhibitors of LTA4 hydrolase. The data has been collected from (20, 24-26).

As one would expect from an affinity label, LTA4 has been shown to inactivate enzymes other than LTA4 hydrolase, i.e. 5-lipoxygenase (21) and platelet-type 12-lipoxygenase (22), for which LTA4 is a product or a substrate, respectively. Recently, it was also shown that leukocyte-type 12 lipoxygenase converted 15S-hydroperoxy-5,8,11,13-eicosatetraenoic acid to 14,15-LTA4, which bound covalently to the enzyme, leading to irreversible inactivation (23). The catalytic apparatus of a lipoxygenase would be expected to be greatly different from that of an epoxide hydrolase. However, binding specificity of LTA4 to lipoxygenases is reasonable, given the structural similarity between LTA4 and arachidonic acid, the natural substrate of these lipoxygenases. Accordingly, the Ki of LTA4 versus 5-lipoxygenase was calculated to ~2 µM, and the Km versus 12-lipoxygenase was determined to ~8 µM. Hence, the affinity between LTA4 and the lipoxygenases appears to be of the same, or even higher, strength as that reported for LTA4 hydrolase (Km in the range of 5-30 µM).

From analysis of mutated forms of LTA4 hydrolase Y378F/Q and E318A, we have now been able to thoroughly investigate the molecular interactions between enzyme and substrate that occur during suicide inactivation of LTA4 hydrolase. Thus, in contrast to what one would expect from a mechanism-based process, we have shown that covalent modification by LTA4 occurred at the active site of a catalytically incompetent enzyme and that a minimal change of an active-site residue generated an enzyme with increased catalytic efficiency and resistance to suicide inactivation.

Taken together, we think that this large body of data is best explained by LTA4 acting as an affinity label. In this model, LTA4 is the natural substrate of the enzyme and at the same time, by virtue of its chemical reactivity, an efficient affinity label. After initial binding of LTA4 to the active site, the hydroxyl group of Tyr-378 may attack the reactive allylic epoxide or a spontaneously formed carbocation thereof to cause covalent modification/enzyme inactivation. Moreover, this mechanistic model agrees well with the current knowledge about LTA4 hydrolase, the kinetics of catalysis and inactivation, the chemistry of LTA4, and, most important, the properties of our mutated enzymes.

    ACKNOWLEDGEMENTS

We express our sincere gratitude to Dr. A. W. Ford-Hutchinson (Merck-Frosst, Canada) for his generous supply of indispensable materials. We are greatly indebted to Ms. Eva Ohlson for excellent technical assistance.

    FOOTNOTES

* This study was supported by funds from the Swedish Medical Research Council (03X-10350 and 03X-217), European Union Grant BMH4-CT96-0229, Stiftelsen Vårdal and Konung Gustav V:s 80-årsfond.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger A recipient of Deutsche Forschungsgemeinschaft fellowship MU 1105/1-1 and Grant MU 1105/2-1). Permanent address: University of Munich, Institute of Pharmaceutical Biology, Karlstr. 29, D-80333 Munich, Germany.

§ To whom correspondence should be addressed. Tel.: 46-8-728 7612; Fax: 46-8-736 0439; E-mail: jesper.haeggstrom@mbb.ki.s.e.

1 The abbreviations used are: LT, leukotriene; LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid; LTB4, 5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid; HPLC, high performance liquid chromatography.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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