Structure, Function, and Regulation of Leukotriene A₄ Hydrolase

JESPER Z. HAEGGSTRÖM

Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, Stockholm, Sweden

	INTRODUCTION

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

Leukotrienes are important chemical mediators in a variety of inflammatory and allergic conditions, including those affecting the respiratory system. These signaling molecules can be divided into two groups: the proinflammatory leukotriene B₄ (LTB₄) and the spasmogenic leukotrienes C₄, D₄, and E₄, also termed cysteinyl-leukotrienes (1). LTB₄ is one of the most powerful chemotactic agents known to date and acts via specific seven-transmembrane, G protein-coupled surface receptors on the target cells (2). The profile of the biological properties of LTB₄ makes it a key component in the complex network of soluble and cell-bound factors that govern the development and maintenance of inflammation. Hence, LTB₄ has been proposed to play a role in a variety of acute and chronic inflammatory diseases such as arthritis, dermatoses, inflammatory bowel disease (IBD), and chronic obstructive pulmonary disease (COPD). In particular, LTB₄ seems to play a role in the recruitment of inflammatory cells to the site of tissue injury. This article gives a brief overview of the biochemistry and molecular biology of LTA₄ hydrolase, the enzyme catalyzing the final step in the biosynthesis of LTB₄.

	LTA4 HYDROLASE IS A KEY ENZYME IN THE 5-LIPOXYGENASE PATHWAY

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

In the biosynthesis of leukotrienes, free arachidonic acid is converted into LTA₄, in two consecutive reactions catalyzed by 5-lipoxygenase. LTA₄ is then stereoselectively hydrolyzed, by LTA₄ hydrolase, into LTB₄. Alternatively, LTA₄ may be conjugated with reduced glutathione (GSH), by a specific S-transferase, to produce LTC₄. Hence, LTA₄ hydrolase catalyzes the most distal step in the biosynthetic pathway leading to the proinflammatory compound LTB₄ (see Figure 2). In the following, the conversion of LTA₄ into LTB₄ is referred to as the epoxide hydrolase activity of the enzyme. Table 1 summarizes the properties of human LTA₄ hydrolase discussed in the present article.

View larger version (53K): [in this window] [in a new window]   Figure 2.   Model of cellular leukotriene biosynthesis. Stimulation of the cell leads to mobilization of Ca2+, which triggers activation and translocation of cytosolic phospholipase A2 (cPLA2) and 5-lipoxygenase (5-LO) to the nuclear envelope. Together with five lipoxygenase-activating protein (FLAP) these enzymes constitute a biosynthetic complex that produces LTA4 for further biosynthesis of LTB4 and LTC4 via the soluble LTA4 hydrolase and membrane-bound LTC4 synthase, respectively.

	LTA4 HYDROLASE IS A BIFUNCTIONAL ZINC METALLOENZYME

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

LTA₄ hydrolase has been purified from a variety of sources as a soluble monomeric protein (for a review see Reference 3). The human, mouse, rat, and guinea pig enzymes have been cloned and sequenced (4). They contain 610 amino acids, excluding the first methionine, and the calculated molecular mass of the human enzyme is about 69,153 Da. The primary structure of LTA₄ hydrolase is unique and bears no resemblance to other known epoxide hydrolases.

Cloning and sequencing of rat kidney aminopeptidase M led to the finding that LTA₄ hydrolase exhibits a weak similarity to this enzyme and several other zinc-containing proteases and peptidases (9). In fact, LTA₄ hydrolase contains a zinc-binding motif (HEXXH-X₁₈ -E), structurally similar to that present in thermolysin, in which the two histidines are the primary ligands and a more distant glutamic acid is the third metal-binding ligand (10). The fourth ligand is always an activated water molecule. From sequence alignments of LTA₄ hydrolase and other members of the same group of metallohydrolases, His-295, His-299, and Glu-318 were identified as potential zinc-binding ligands (see below).

	LTA4 HYDROLASE CONTAINS ONE CATALYTIC ZINC

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The discovery of the zinc-binding signature in LTA₄ hydrolase prompted us and others to investigate the possible zinc content of the enzyme (11, 12). For this purpose, we analyzed purified LTA₄ hydrolase from human leukocytes by atomic absorption spectrometry, which revealed the presence of one zinc atom per enzyme molecule. Furthermore, the chelating agent 1,10-phenanthroline inactivated LTA₄ hydrolase by removing the zinc, thus generating the apoenzyme of LTA₄ hydrolase. However, addition of stoichiometric amounts of zinc to the apoenzyme restored the enzyme activity and this effect reached a plateau at a 1:1 molar ratio of metal versus protein, i.e. at the same metal-to-protein stoichiometry as in the native holoenzyme. Hence, the primary function of the metal seems to be catalytic rather than structural (Figure 1).

View larger version (43K): [in this window] [in a new window]   Figure 1.   Structural and functional elements of LTA4 hydrolase, a bifunctional zinc metalloenzyme. Ala-4-NA = alanine 4-nitroanilide (the peptidase substrate). For details see text.

	LTA4 HYDROLASE POSSESSES AN INTRINSIC AMINOPEPTIDASE ACTIVITY

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

The identification of LTA₄ hydrolase as a member of a family of zinc metalloproteases suggested that it also possessed a peptide-cleaving activity, in addition to its well-characterized epoxide hydrolase activity (conversion of LTA₄ into LTB₄). This was indeed the case and LTA₄ hydrolase was shown to hydrolyze a number of synthetic chromogenic substrates (12, 13) (Figure 1).

LTA₄ hydrolase belongs to the M1 class of metallohydrolases, all of which contain a similar zinc signature, and other enzymes in this group include aminopeptidase M (> 20% identity) and aminopeptidase B (> 30% identity). It appears that the presence of a zinc-binding motif in a protein predicts only a peptide-cleaving activity. Thus, preparations of aminopeptidase M from porcine and hog kidney, as well as bovine intestine, failed to convert LTA₄ into LTB₄ (12, 13). Furthermore, an aminopeptidase from Caenorhabditis elegans, > 40% identical (at the amino acid level) with mammalian LTA₄ hydrolase, was cloned and characterized (14). This enzyme also lacked an epoxide hydrolase activity.

Alignment of the primary structures of LTA₄ hydrolase and aminopeptidase B shows that they are > 80% identical over > 70 residues, including the zinc signatures. The functional significance of this segment of the proteins is uncertain but may be related to the substrate specificity of the peptidase activity; both enzymes prefer arginyl peptides (see below). Moreover, it has been reported that aminopeptidase B exhibits a weak epoxide hydrolase activity (15).

Chloride Stimulation of the Peptidase Activity via a Putative Anion-binding Site

The peptidase activity is greatly stimulated by certain monovalent anions, in particular chloride ions and thiocyanate ions (Figure 1). From kinetic data of the chloride stimulation, we found that the effect obeyed saturation kinetics. An apparent affinity constant for chloride was calculated as ~ 100 mM, which is close to the extracellular concentration of this electrolyte (16). Thus, chloride stimulation of LTA₄ hydrolase appears to be mediated via an anion-binding site. In contrast to the effects on the peptidase activity, no chloride stimulation was observed for the epoxide hydrolase activity. Considering the differences in chloride concentration between the intracellular and extracellular compartments, one can speculate that the peptidase activity of LTA₄ hydrolase may proceed only outside the cell, whereas the epoxide hydrolase activity may operate on either side of the cell membrane. Notably, LTA₄ hydrolase activity has been detected in the plasma of several mammals and high levels of the enzyme have been found in human bronchoalveolar lavage fluid (17, 18). An extracellular role for the peptidase activity is also supported by the finding that albumin, the major protein constituent of plasma, can stimulate the peptidase activity of LTA₄ hydrolase (19).

Zinc and Other Divalent Cations Inhibit LTA₄ Hydrolase

Both enzyme activities of LTA₄ hydrolase are reversibly inhibited by zinc in a dose-dependent manner, with median inhibitory concentration (IC₅₀) values of 10 and 0.1 µM for the epoxide hydrolase and peptidase activity, respectively (20). Divalent cations other than zinc also inhibit LTA₄ hydrolase with different specificity and potency for the two enzyme activities. For instance, Mn²⁺ and Co²⁺, which inhibited the peptidase activity with IC₅₀ values < 10 µM, had no effect on the conversion of LTA₄ into LTB₄ at 1 mM concentration (20). The mechanism of zinc inhibition of LTA₄ hydrolase is presently unknown.

	SUBSTRATE SPECIFICITY OF LTA4 HYDROLASE

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

The epoxide hydrolase activity of LTA₄ hydrolase exhibits a narrow substrate specificity and accepts only a 5,6-trans-epoxide with a free carboxylic acid at C-1 of the fatty acid (21). Also, the double-bond geometry of the substrate is essential for catalysis. In fact, besides LTA₄, the double-bond isomers LTA₃ and LTA₅ are the only other accepted, albeit weak, substrates known to date (21). Moreover, all these leukotriene epoxides inactivate LTA₄ hydrolase during catalysis, a phenomenon referred to as suicide inactivation (see below).

In contrast, the active site corresponding to the peptidase activity appears to be promiscuous. Thus, an array of different synthetic substrates is cleaved by LTA₄ hydrolase, including nitroanilide and 2-naphthylamide derivatives of various amino acids, in particular alanine and arginine (12, 13, 24). Interestingly, the enzyme has been shown to cleave the N-terminal arginine from several tripeptides with high efficiency. For instance, the peptides Arg-Gly-Asp, Arg-Gly-Gly, and Arg-His-Phe were hydrolyzed with specificity constants (k_cat/K_m) in the same order of magnitude as for hydrolysis of LTA₄, suggesting that LTA₄ hydrolase is an arginyl tripeptidase (24). Notably, the endogenous substrate(s) for the peptidase activity of LTA₄ hydrolase still remains to be identified.

	CELLULAR AND SUBCELLULAR LOCALIZATION

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

In all species investigated thus far, LTA₄ hydrolase has been widely spread among organs, tissues, and individual cell types (for a review see Reference 3). Studies in guinea pig, rat, and humans have shown that the enzyme is particularly abundant in the intestine, spleen, lung, and kidney. Also, it was reported that Xenopus laevis expresses high levels of LTA₄ hydrolase activity in the reproductive organs (25). In the blood, neutrophils, monocytes, lymphocytes, and erythrocytes are rich sources of LTA₄ hydrolase. In contrast, eosinophils have low levels and basophils and platelets seem to be virtually devoid of the enzyme. With respect to the lung, epithelial cells and alveolar macrophages appear to express high levels of LTA₄ hydrolase, whereas mast cells seem to have small amounts of the enzyme. Considering the fact that leukotriene biosynthesis is generally regarded as a process restricted to the white blood cells and bone marrow, the broad distribution of LTA₄ hydrolase has been difficult to rationalize from a functional point of view. One explanation has been so-called transcellular biosynthesis, a phenomenon in which a given intermediate is exported from a donor cell to a recipient cell for further metabolism (see below).

On purification, LTA₄ hydrolase is consistently recovered as a soluble protein and therefore it is generally assumed that the activity resides in the cytosol. In one study, a membrane-bound activity in liver cells was reported (26). Specific organelles such as nuclei, mitochondria, or peroxisomes have seldom, if ever, been examined as potential sources of a soluble enzyme activity. As discussed by Peters-Golden in a separate chapter of this volume, all key enzymes in the leukotriene biosynthesis will assemble at the nuclear envelope on cellular activation (Figure 2). Notably, LTA₄ hydrolase seems to be the only exception to this rule, since migration to the nuclear membrane in response to cell stimulation has not been demonstrated.

	GENE STRUCTURE AND REGULATION

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

The human LTA₄ hydrolase gene is > 35 kbp, exists in a single copy, and contains 19 exons ranging in size from only 24 to 312 bp (27). Fluorescence in situ hybridization has been used to map the gene to chromosome 12q22. The putative promoter region (~ 4 kbp upstream of the transcription initiation site) contained a phorbol ester response element (AP2) and two xenobiotic-response elements (XRE) but no definitive TATA box (see Table 1). Further upstream of these elements, approximately 2.2 kbp from the initial methionine, there is also a consensus sequence for an Alu repeat, which may participate in gene regulation (28).

Sequential reverse transcriptase polymerase chain reaction (RT-PCR) mapping has been used to identify a short transcript of the LTA₄ hydrolase gene (29). This mRNA is formed by alternative splicing of an 83-bp exon (exon 17), which in turn leads to an altered reading frame and a preterminal stop after 22 amino acids. As a consequence, this short mRNA predicts the expression of an LTA₄ hydrolase isoform with a calculated molecular mass of 59 kD and a distinct C terminus. However, whether this isoform is enzymatically active or even expressed in vivo, remains to be determined.

	LTA4 HYDROLASE IN TRANSCELLULAR BIOSYNTHESIS OF LTB4

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

Transcellular metabolism means that a certain endogenous compound is exported by a donor cell to a recipient cell for further conversion(s) into metabolites that sometimes none of the participating cell types can generate themselves. This route of biosynthesis has been described for a number of different lipid mediators and in particular for LTA₄ (30). Thus, activated leukocytes can generate LTA₄, which can be metabolized into LTB₄ and LTC₄ as well as lipoxins. As mentioned above, LTA₄ hydrolase has a widespread distribution, and is present in many cell types that lack significant 5-lipoxygenase activity and thus the ability to generate the substrate LTA₄, e.g., endothelial cells, erythrocytes, and lymphocytes (Figure 3). This uneven distribution of two intimately coupled enzyme activities, i.e., LTA₄ hydrolase and 5-lipoxygenase, has been explained in terms of transcellular biosynthesis, and indeed transfer of LTA₄ from activated leukocytes to a variety of other cell types has been demonstrated in vitro, a phenomenon that is promoted by tight cell-cell interactions (31). With respect to the respiratory system, several studies have indicated that LTB₄ biosynthesis is amplified by transcellular mechanisms involving activated neutrophils as the donor cell and airway epithelial cells or alveolar macrophages as the recipients of LTA₄ (32, 33). In addition, LTA₄ hydrolase is present at high levels in bronchoalveolar lavage fluid and can convert neutrophil-derived LTA₄ into LTB₄ (18). Inasmuch as LTA₄ hydrolase is a bifunctional enzyme, it is possible that its previously unknown peptide-cleaving activity accounts for the disproportionate distribution of 5-lipoxygenase and LTA₄ hydrolase. Hence, it is possible that the epoxide hydrolase activity is confined to various types of leukocytes, whereas the peptidase activity may predominate in cells not involved in leukotriene biosynthesis or in the extracellular compartment, as discussed above.

View larger version (46K): [in this window] [in a new window]   Figure 3.   Transcellular biosynthesis of LTB4. Illustrated here are the potential routes for intravascular amplification of LTB4 biosynthesis by transcellular metabolism of LTA4. Solid arrows indicate release of LTA4 (and LTB4) from activated monocytes (1) or neutrophils (2). Dashed arrows indicate various routes for metabolism of LTA4 into LTB4 via lymphocytes (3), erythrocytes (4), or endothelial cells (5).

	SYNTHETIC INHIBITORS OF LTA4 HYDROLASE

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

Before the identification of LTA₄ hydrolase as a zinc metalloenzyme with a peptide-cleaving activity, no reasonably potent or specific enzyme inhibitors were known. An immediate consequence of the homology to zinc proteases was to test the effect of a number of peptidase inhibitors. As a result, bestatin and captopril, classic inhibitors of aminopeptidases and angiotensin-converting enzyme, respectively, were found to be effective inhibitors of LTA₄ hydrolase (34). Later, work in several laboratories, led to the development of more powerful and selective compounds. Thus, on the basis of proposed reaction mechanisms and inhibitor-enzyme interactions for other zinc hydrolases, new compounds were synthesized and tested for their effect on the epoxide hydrolase and peptidase activity of LTA₄ hydrolase. Among more than 30 different structures, we found 2 compounds, an -keto--amino ester and a thioamine, which were potent tight-binding inhibitors with IC₅₀ values in the low micromolar to nanomolar range (35, 36). In addition, a series of -amino hydroxylamine and amino hydroxamic acids were developed, among which one hydroxamate turned out to be as potent as the above-mentioned substances (37). Moreover, the -keto--amino ester, the thioamine, and the hydroxamate are all potent and selective inhibitors of LTB₄ biosynthesis in intact human leukocytes (36, 37). Furthermore, a class of -[(-arylalkyl)aryl]alkanoic acids was reported to inhibit LTA₄ hydrolase in the low micromolar range, one of which was metabolically stable after oral administration to rats (38). Perhaps the most interesting and promising inhibitors of LTA₄ hydrolase were presented by Searle. Particularly SC-57461, N-methyl-N-[3-[4-(phenylmethyl)-phenoxy]propyl]--alanine, blocked ionophore-induced LTB₄ production in human whole blood with an IC₅₀ of only 49 nM (39), was orally active, and showed promising results in an animal model of colitis (40).

	IDENTIFICATION OF CATALYTICALLY IMPORTANT AMINO ACIDS IN LTA4 HYDROLASE

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

The crystal structure of LTA₄ hydrolase has not yet been determined and, hence, no detailed molecular information about the active site is available. Nevertheless, we have used a combination of computer-assisted sequence comparisons, site-directed mutagenesis, and biochemical analysis to identify functionally important regions and amino acids in LTA₄ hydrolase (Table 1).

His-295, His-299, and Glu-318 are the Zinc-binding Ligands in LTA₄ Hydrolase

The three proposed zinc-binding ligands in LTA₄ hydrolase, His-295, His-299, and Glu-318, were individually replaced by tyrosine, tyrosine, and glutamate, respectively, none of which can bind zinc (41). The three mutants [H295Y]-, [H299Y]-, and [E318Q]LTA₄ hydrolase (single-letter code for the amino acid change) were all found to be devoid of both the epoxide hydrolase and peptidase activities as well as the prosthetic zinc. The concomitant loss of both enzyme activities and zinc content in all three mutants confirmed the identity of the predicted zinc-binding ligands and underscored the critical role of the zinc atom in both the epoxide hydrolase and peptidase reactions (Figure 1).

Selective Abrogation of the Peptidase Activity by Mutation of Glu 296

A conserved glutamic acid, juxtaposed to the first zinc-binding ligand, is a typical feature of the zinc signature of many zinc proteases, e.g., thermolysin, neutral endopeptidase, and aminopeptidase M, and is believed to take part in the proteolytic reaction. In LTA₄ hydrolase, Glu-296 occupies this position in the zinc-binding motif (10). To investigate the role of Glu-296 in the two catalytic activities of LTA₄ hydrolase, we constructed the two mutants [E296Q]- and [E296A]LTA₄ hydrolase, which were purified to homogeneity to allow metal analyses and enzyme activity determinations (42). Both mutants contained the expected amounts of zinc (~ 1 Eq) and exhibited significant epoxide hydrolase activity. In contrast, the two mutated enzymes were virtually inactive with respect to the peptidase activity. Apparently, we had been able to selectively abrogate one of the two catalytic activities of LTA₄ hydrolase. Hence, Glu-296 is essential only for the peptidase activity, presumably as a general base (see below). Moreover, the selective effect of mutation on only one of the two activities shows that the corresponding active site(s) are overlapping (Figure 1).

Tyr-383 is a Potential Proton Donor in the Peptidase Reaction

In addition to the zinc-binding motif, computer-assisted sequence comparisons between LTA₄ hydrolase and aminopeptidase M revealed the presence of a conserved nonapeptide centered around a tyrosine residue (43). This short peptide has been suggested to represent a proton donor motif and in LTA₄ hydrolase, Tyr-383 was proposed to be the proton-donating residue (43). To detail the role of Tyr-383 in the two catalytic activities of LTA₄ hydrolase, we exchanged this residue for a phenylalanine, histidine, or glutamine residue by site-directed mutagenesis (44). Again, the mutated enzymes were purified to apparent homogeneity to allow enzyme activity determinations. All mutants exhibited significant albeit substantially reduced epoxide hydrolase activities. In contrast, none of them displayed any significant peptidase activity against alanine-4-nitroanilide. Thus, these results are in agreement with a role for Tyr-383 in the peptidase reaction, perhaps as a proton donor (Figure 1).

	MUTANTS OF Tyr-383 CAN PRODUCE BOTH LTB4 AND 5S,6S-DHETE

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

A closer investigation of the catalytic properties of the mutants of Tyr-383, in particular [Y383Q]LTA₄ hydrolase, revealed that they could convert LTA₄, not only into LTB₄, but also into a novel enzymatic product structurally identified as 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid (5S,6S-DHETE) (45). The stereochemistry of the vicinal diol, i.e., 5S, 6S, in combination with the finding that the nucleophilic attack of water is directed toward C-6, allows us to conclude that the hydrolysis must occur according to an S_N1 mechanism, which by definition involves a carbocation intermediate. Of note, since the mutants could produce both LTB₄ and 5S,6S-DHETE, it seems likely that conversion of LTA₄ into LTB₄ follows the same reaction mechanism.

	PUTATIVE REACTION MECHANISMS FOR THE PEPTIDASE AND EPOXIDE HYDROLASE ACTIVITY

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

On the basis of X-ray crystallographic data, two reaction mechanisms have been discussed for the proteolytic activity of thermolysin, which contains a zinc site structurally similar to that of LTA₄ hydrolase. Thus, the conserved Glu-143 has been suggested to act as a general base or to form an anhydride with the substrate (46, 47). In the former and most favored mechanism, a water is displaced from the zinc atom by the carbonyl oxygen of the substrate and then polarized by the carboxylate of the glutamic acid to promote an attack on the carbonyl carbon of the scissile peptide bond. Simultaneously, a proton is transferred to the nitrogen of the peptide bond from an adjacent amino acid. Considering the results obtained by mutational analysis (see above), Glu-296 and Tyr-383 would correspond to the base and proton donor in LTA₄ hydrolase, respectively.

In our work with inhibitors of LTA₄ hydrolase, a hydroxamic acid was developed with strong potency. This molecule has a bicyclic portion probably buried in a hydrophobic pocket of the protein, a hydroxamate moiety that chelates the zinc and a spacer with an ultimate carboxylate, presumably binding to a site that normally recognizes the carboxylate of the substrate (48). If the structure of the inhibitor is superimposed onto the structure of LTA₄, the hydroxamate gets close to the epoxide, suggesting that the epoxide moiety is close to the zinc. In keeping with the evidence of an S_N1 reaction, one may speculate that the metal acts as a Lewis acid to activate and open the labile allylic epoxide (48). This in turn generates a carbocation intermediate, the charge of which is delocalized over the triene system. In the final reaction step, water will be introduced at C-12 in an enzyme-directed stereoselective manner probably involving a base. Mechanistically, this scheme has many features in common with nonenzymatic hydrolysis of LTA₄ into 6-trans- and 12-epi-6-trans-LTB₄. Apparently, LTA₄ hydrolase is responsible for the formation of a ⁶-cis-⁸- trans-¹⁰-trans geometry of the conjugated triene as well as a stereoselective introduction of H₂O at C-12 to generate a hydroxyl group in R configuration.

	MOLECULAR MECHANISM FOR SUICIDE INACTIVATION OF LTA4 HYDROLASE

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

Typically, LTA₄ hydrolase is covalently modified and inactivated by its endogenous lipid substrate LTA₄, a process commonly referred to as suicide inactivation. This built-in mechanism for enzyme inhibition may be of importance for the overall regulation of LTB₄ biosynthesis in vivo, and Fitzpatrick and coworkers have suggested that suicide inactivation is a mechanism-based process (49, 50). For instance, they have shown that the epoxide hydrolase and peptidase activities are lost simultaneously and irreversibly in a time-dependent, saturable process that is of pseudo-first-order kinetics and dependent on catalysis. Active site specificity has been demonstrated by protection with competitive inhibitors and mass spectrometric analysis has revealed that suicide inactivation occurs predominantly in a 1:1 stoichiometry between lipid and protein.

Identification of a Peptide Region Involved in Suicide Inactivation

We used differential lysine-specific peptide mapping of untreated and suicide-inactivated enzyme to identify a peptide to which LTA₄ binds during suicide inactivation (51). Amino acid sequence analysis showed that this peptide spans 21 residues from Leu-365 to Lys-385. Owing to the number of amino acids and the fact that it originates from a Lys-C digest, the peptide was denoted K21. Interestingly, Tyr-383, the previously discussed proton donor of the peptidase reaction, was located within peptide K21, supporting the conclusion that Tyr-383 is an active site residue (Figure 1). When LTA₄ methyl and ethyl ester were used as suicide inhibitors we produced results equivalent to those described above. Peptide mapping of enzyme treated with LTA₄ ethyl ester allowed isolation of a modified peptide in amounts sufficient for complete Edman degradation. The sequence was identical to that of K21, with the exception of a gap corresponding to Tyr-378 of intact LTA₄ hydrolase, demonstrating that LTA₄ ethyl ester had bound to this residue during suicide inactivation of LTA₄ hydrolase.

Tyr-378 Is a Major Structural Determinant for Suicide Inactivation

To study the role of Tyr-378 in suicide inactivation and its potential catalytic function, this residue was exchanged for phenylalanine or glutamine in two separate mutants (52). In addition, each of two adjacent and potentially reactive residues, Ser-379 and Ser-380, was exchanged for alanine. Interestingly, wild-type enzyme and the mutants [S379A]- and [S380A] LTA₄ hydrolase were equally susceptible to suicide inactivation whereas the enzymes mutated at position 378 were no longer inactivated or covalently modified by LTA₄. Furthermore, for [Y378F]LTA₄ hydrolase, the value of k_cat for epoxide hydrolysis was increased 2.5-fold over that of the wild-type enzyme. Thus, by a single point mutation in LTA₄ hydrolase, catalysis and covalent modification/inactivation had been dissociated, yielding an enzyme with increased turnover and resistance to mechanism-based inactivation.

A more detailed examination of the catalytic properties of [Y378F]- and [Y378Q]LTA₄ hydrolase revealed that they were able to generate not only LTB₄ but also a second metabolite of LTA₄, in a yield of about 20-30% (53). On the basis of physicochemical analyses and comparison with a synthetic standard the novel metabolite was assigned the tentative structure 5S,12R-dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e., ⁶-trans-⁸-cis-LTB₄. Hence, Tyr-378 appears to be involved in the positioning of the cis double bond in the product, perhaps by assisting in the proper alignment of LTA₄ in the substrate-binding pocket or by promoting a favorable conformation of a putative carbocation intermediate (Figure 1).

Inactivation and Covalent Modification Occurs via an Affinity-labeling Mechanism

As discussed above, suicide inactivation has previously been rationalized as a mechanism-based process (49, 50). However, during our work with mutated forms of LTA₄ hydrolase we found that the mutant [E318A]LTA₄ hydrolase, which lacks the prosthetic zinc and is therefore catalytically incompetent, was covalently modified by LTA₄ (54). The degree of modification was even greater than for the wild-type enzyme. Furthermore, Tyr-378, the major site of attachment between LTA₄ and the protein, is a catalytically nonessential residue, since [Y378F]LTA₄ hydrolase exhibits increased substrate turnover and resistance to inactivation by LTA₄. Apparently, inactivation and or covalent modification can be completely dissociated from catalysis.

These data are 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 also agrees well with the current knowledge about LTA₄ hydrolase, the kinetics of catalysis and inactivation, and the chemistry of LTA₄.

	Footnotes

Correspondence and requests for reprints should be addressed to Jesper Z. Haeggström, M.D., Ph.D., Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171-77 Stockholm, Sweden. E-mail: jesper.haeggstrom{at}mbb.ki.se

Acknowledgments: The author is most grateful to Anders Wetterholm, Juan F. Medina, Martin J. Mueller, Martina Andberg, Filippa Strömberg-Kull, Eva Ohlson, and Bengt Samuelsson for their contributions to the studies described in this article.

Supported by the Swedish Medical Research Council (O3X-10350), the European Union (BMH4-CT960229), and Konung Gustav V:s 80-Årsfond.

	References

TOP INTRODUCTION LTA4 HYDROLASE IS A... LTA4 HYDROLASE IS A... LTA4 HYDROLASE CONTAINS ONE... LTA4 HYDROLASE POSSESSES AN... SUBSTRATE SPECIFICITY OF LTA4... CELLULAR AND SUBCELLULAR... GENE STRUCTURE AND REGULATION LTA4 HYDROLASE IN TRANSCELLULAR... SYNTHETIC INHIBITORS OF LTA4... IDENTIFICATION OF CATALYTICALLY... MUTANTS OF Tyr-383 CAN... PUTATIVE REACTION MECHANISMS... MOLECULAR MECHANISM FOR SUICIDE... REFERENCES

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