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Leukotriene A4 hydrolase: identification of a common carboxylate recognition site

for the epoxide hydrolase and aminopeptidase substrates

Peter C. Rudberg1*, Fredrik Tholander1*, Martina Andberg1, Marjolein M.G.M. Thunnissen2

and Jesper Z. Haeggström1§

1 Department of Medical Biochemistry and Biophysics, Division of Chemistry II,

Karolinska Institutet, S-171 77 Stockholm, Sweden.

2 Department of Molecular Biophysics, Kemicentrum, Lund University, S-221 00 Lund,

Sweden.

* These authors contributed equally to the present study

§ To whom correspondence should be addressed:

Tel +46-8-728 76 12, Fax +46-8-736 04 39, E-Mail: jesper.haeggstrom@mbb.ki.se

Running title: A dual carboxylate recognition site in LTA4 hydrolase

JBC Papers in Press. Published on April 12, 2004 as Manuscript M401031200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

Leukotriene (LT) A4 hydrolase is a bifunctional zinc metalloenzyme, which converts LTA4

into the neutrophil chemoattractant LTB4 and also exhibits an anion-dependent

aminopeptidase activity. In the X-ray crystal structure of LTA4 hydrolase, Arg563 and

Lys565 are found at the entrance of the active center. Here we report that replacement of

Arg563, but not Lys565, leads to complete abrogation of the epoxide hydrolase activity.

However, mutations of Arg563 do not seem to affect substrate binding strength, since values

of Ki for LTA4 are almost identical for wild type and [R563K]LTA4 hydrolase. These results

are supported by the 2.3 Å crystal structure of [R563A]LTA4 hydrolase, which does not

reveal structural changes that can explain the complete loss of enzyme function. For the

aminopeptidase reaction, mutations of Arg563 reduce the catalytic activity (Vmax=0.3-20%),

whereas mutations of Lys565 have limited effect on catalysis (Vmax =58-108%). However, in

[K565A]- and [K565M]LTA4 hydrolase, i.e. mutants lacking a positive charge, values of the

Michaelis constant for alanine-p-nitroanilide increase significantly (Km=480-640%).

Together, our data indicate that Arg563 plays an unexpected, critical role in the epoxide

hydrolase reaction, presumably in the positioning of the carboxylate tail to ensure perfect

substrate alignment along the catalytic elements of the active site. In the aminopeptidase

reaction, Arg563 and Lys565 seem to cooperate to provide sufficient binding strength and

productive alignment of the substrate. In conclusion, Arg563 and Lys565 possess distinct

roles as carboxylate recognition sites for two chemically different substrates, each of which is

turned over in separate enzymatic reactions catalyzed by LTA4 hydrolase.

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Introduction

The leukotrienes (LTs) are a class of structurally related lipid mediators involved in the

development and maintenance of inflammatory and allergic reactions (1,2). In the

biosynthesis of LTs, 5-lipoxygenase converts arachidonic acid into the unstable epoxide

LTA4. This intermediate may in turn be conjugated with glutathione to form the spasmogenic

LTC4, or hydrolyzed into the proinflammatory lipid mediator LTB4, in a reaction catalyzed by

LTA4 hydrolase (LTA4H). Leukotriene B4 is a classical chemoattractant of human neutrophils

and triggers adherence and aggregation of leukocytes to vascular endothelium at only

nanomolar concentrations (3). In addition, LTB4 modulates immune responses (4,5) and

recent studies show that this lipid mediator is involved in early effector CD4+ and CD8+ T cell

recruitment to sites of inflammation (6-8). Moreover, LTB4 participates in the host-defense

against infections (9,10) and is a key mediator of PAF-induced lethal shock (11-13). These

effects are signaled via specific, G-protein coupled receptors for LTB4 (BLT1 and BLT2)

(14,15). The relative contributions of these two receptors to signaling and bioactivity are

presently not clear.

LTA4H (EC 3.3.2.6) is a bifunctional zinc metalloenzyme, exhibiting an anion-

dependant aminopeptidase activity in addition to its epoxide hydrolase activity, i.e. the

hydrolysis of the unstable allylic epoxide LTA4 into LTB4, for a review see (16). Unlike the

aminopeptidase activity, the epoxide hydrolase activity is restrained by suicide inactivation

which involves binding of LTA4 to Tyr378, which is located within a 21-residue active-site

peptide denoted K21 (17,18). The epoxide hydrolase reaction of LTA4H is also unique in the

sense that the stereoselective introduction of water to the carbon backbone of LTA4 occurs

several methylene units away from the epoxide moiety, presumably proceeding via a

carbocation intermediate (19). Furthermore, Asp375 was shown to assist in the introduction of

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the 12R hydroxyl group of LTB4 (20), a key component for the biologic activity of LTB4 (21-

23).

Certain arginyl di- and tripeptides as well as p-nitroanilide derivatives of Ala and Arg

are very efficient substrates for the aminopeptidase activity (24). Although it has never been

proven, it is generally believed that the peptidase activity is involved in the processing of

bioactive peptides related to inflammation and host-defense. Several lines of evidence

indicate that the aminopeptidase activity follows a zinc-assisted general base mechanism with

Glu296 and Tyr383 acting as the general base and proton donor, respectively. Moreover, in

recent studies, Glu271 was identified as the recognition site for the N-terminal amino group

of the peptidase substrate (25). Recently, we solved the crystal structure of LTA4H in

complex with the aminopeptidase inhibitor bestatin at 1.95 Å resolution, which has given

important clues to the molecular mechanisms of the two enzyme reactions (26).

Earlier work with chemical modification already indicated the presence of essential Arg

residues near or at the active center of LTA4H (27). From the X-ray crystal structure of

LTA4H, a positively charged site was identified in a cavity formed between the N-terminal,

the central catalytic, and the C-terminal domain (Fig. 1). This site, composed of Arg563 and

Lys565, is located in a wide portion of the cavity, near its entrance, and in the vicinity of the

carboxyl group of bestatin. These two residues are positioned in the first turn of an α-helix

pointing towards the active site. While Arg563 is buried, Lys565 is fully exposed to solvent.

Also, when LTA4 was modeled into the active site, electrostatic interactions seemed

possible between the C1 carboxylate of LTA4 and the positively charged side groups of

Arg563 and/or Lys565 (Fig. 1). Furthermore, in the crystal structure of LTA4H in complex

with a specific hydroxamic acid inhibitor that is a structural mimic of LTA4, a direct

interaction between the carboxylic moiety of this inhibitor and Arg563 is seen (28). In this

report we used site-directed mutagenesis combined with X-ray crystallography and inhibition

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studies to analyze the functional role of Arg563 and Lys565 as putative carboxylate

recognition sites in LTA4H. We show that Arg563 is required for the epoxide hydrolase

activity, presumably in an indirect manner to achieve correct alignment of LTA4 along the

catalytic elements of the active site. As a result, Arg563 is a key residue during LTB4

formation. For the aminopeptidase reaction, we propose that Lys565 assists Arg563 for

optimal substrate binding. Thus, we have identified a common carboxylate recognition site for

lipid and peptide substrates in the active center of LTA4H.

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Experimental procedures

Materials

Oligonucleotides were synthesized by Cybergene, Stockholm, Sweden. LTA4 methyl ester

was synthesized as described (29) or purchased from BIOMOL Res. Lab, Plymouth Meeting,

PA, USA. LTA4 methyl ester was saponified in tetrahydrofuran with 1M LiOH (6% v/v) for

48 h at 4ºC. Alanine-p-nitroanilide, isopropyl-β-D-thiogalactopyranoside,

phenylmethylsulfonyl fluoride, soybean trypsin inhibitor and streptomycin sulphate were

purchased from Sigma, Sweden. Nickel nitrilo-triacetic-acid resin was from QIAGEN.

Mutagenesis of human LTA4H cDNA

Site-directed mutagenesis was carried out by PCR on the recombinant plasmid pT3MB5 for

expression of (His)6-tagged LTA4H in E. coli. This plasmid is a variant of pT3MB4 (18) in

which an Nsi I restriction site has been eliminated by a single nucleotide exchange. Briefly,

site-directed mutagenesis involves two consecutive steps of PCR comprising four different

primers, according to the megaprimer method (30). Polymerase chain reactions were carried

out in a total volume of 50 µl, using 1x Pfu Polymerase Buffer, 125-150 ng each of primers A

and B, 1 U Pfu polymerase, 10 nmol each of dNTPs and 100 ng template. In the second

reaction, 15-20 µl of the first PCR mix was used to supply the reaction with the megaprimer,

which was used together with a primer C (125-150 ng). The amplification program included

an initial round of denaturation at 94°C (60 s), annealing at 55-66°C (60 s) and elongation at

72°C (90 s), followed by 30 cycles of denaturation (45 s), annealing (30 s) and elongation (60

s) on a PE GeneAmp PCR System 2400.

For generation of [E560Q]-, [R563K]-, [R563A]-, [R563M]-, [K565R]-, [K565A]-,

[K565M]- and [R563K/K565R]LTA4H, a Bfr I and a Nsi I site were used. DNA fragments

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were cleaved with Bfr I/Nsi I and purified by agarose gel electrophoresis (1.5 %) followed by

extraction (QIAEX II Gel Extraction Kit). Mutated fragments were ligated into pT3MB5 (T4

DNA Ligase Protocol), opened with the same restriction enzymes. Competent E. coli cells

(JM101) were transformed with mutated recombinant plasmid and grown in LB medium

containing ampicillin (100 µg/ml). Stock cultures were kept at -70°C in a 1:1 mixture of

culture medium and 40 % (v/v) glycerol/0.75 % (w/v) NaCl, respectively. Recombinant

plasmids were purified using Wizard Minipreps Plus and the entire mutated inserts were all

sequenced using Dyenamic ET Terminator Cycle Sequencing Kit (Amersham Pharmacia

Biotech) to confirm that no nucleotide alterations had occurred in addition to the desired

mutation.

Protein expression and purification

Mutated enzymes were expressed as N-terminal (His)6-tag fusion proteins in E. coli (JM101)

cells grown at 37°C in M9 medium (50 mM Na2HPO4, 22 mM KH2PO4, 20 mM NH4Cl, 8.5

mM NaCl), pH 7.4, containing 0.4 % glucose (w/v), 0.2 % (w/v) casamino acids, 2 mM

MgSO4 and 0.1 mM CaCl2. At OD620 ≈ 0.2, isopropyl-β-D-thiogalactopyranoside was added

to a final concentration of 500 µM. Cells were harvested at OD620 ≈ 1.8, pelleted at 1000 x g

and resuspended in 30 ml homogenization buffer (50 mM Tris-HCl, pH 8.0, containing

soybean trypsin inhibitor) supplemented with phenylmethylsulfonyl fluoride (1 mM). Nucleic

acids were removed by streptomycin sulphate precipitation. After centrifugation (10 000 x g

for 15 min), the supernatant was filtered (0.22 µm) and applied to a nickel nitrilo-triacetic-

acid resin. The column was washed with 1 bed volume of 50 mM Tris-HCl, pH 8.0, 50 mM

sodium phosphate buffer, pH 6.8, containing 0.5 M NaCl, and 50 mM Tris-HCl, pH 8.0, with

each solution supplemented with 10 mM imidazole. The His-tagged protein was eluted with

1.4 bed volumes of 50 mM Tris-HCl, pH 8.0, containing 100 mM imidazole. The purity of the

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final preparation was assessed by SDS-PAGE on a Pharmacia Phast system, using 10-15 %

gradient gels, subsequently stained with Coomassie brilliant blue. Protein concentrations were

determined by the Bradford method using a MCC/340 96-well multiscan spectrophotometer

and BSA as standard (31).

Aminopeptidase activity assays

The aminopeptidase activity, expressed as Vmax, was determined in a spectrophotometric assay

at 405 nm, using a MCC/340 multiscan spectrophotometer, essentially as described (32).

Briefly, the enzyme (1-20 µg) was incubated at room temperature in 96-well microtiter plates

with alanine-p-nitroanilide as substrate in 50 mM Tris-HCl, pH 8.0, containing 100 mM KCl.

The absorbance at 405 nm was measured at 10 min intervals. Different concentrations of

substrate (0.125, 0.25, 0.5, 1, 2, 4, 8 mM) were used for kinetic determinations. Spontaneous

hydrolysis of the substrate was corrected for by subtracting the absorbance of blank

incubations without enzyme. The Michaelis constant (Km) and Vmax were determined by

plotting the calculated velocity as a function of substrate concentration, whereby a non-linear

regression analysis was performed.

Epoxide hydrolase activity assays and reverse-phase HPLC

The epoxide hydrolase activity, expressed as Vmax, was determined from incubations of

enzyme (1-20 µg) in 100 µl 10 mM Tris-HCl, pH 8.0, with LTA4 (2.5-125 µM) for 30 s on

ice. Reactions were quenched with 200 µl of MeOH, followed by the addition of 0.4 nmol of

prostaglandin B1 (PGB1) or PGB2 as internal standard. Samples were acidified with 5 µl of

acetic acid (10 %), and metabolites were extracted on solid phase Chromabond C18 columns.

Metabolites of LTA4 were separated by isocratic reverse-phase HPLC on a Waters Nova-Pak

C18 column eluted with a mixture of methanol/acetonitrile/water/acetic acid (30:30:40:0.01

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by vol.) at a flow rate of 1.2 ml/min. The UV detector was set at 270 nm and metabolites were

quantified using Chromatography Station for Windows version 1.7 computer software.

Calculations were based on peak area measurements and the known extinction coefficients for

the internal standards PGB1 and PGB2 (30 000 M-1 x cm-1) as well as LTB4 (50 000 M-1 x cm-

1). The Michaelis constant (Km) and Vmax were determined by plotting the calculated velocity

as a function of substrate concentration, whereby a non-linear regression analysis was

performed.

Inhibition assays

Prior to performing aminopeptidase activity assays, wild type-, [R563K]- and

[R563M]LTA4H were pre-incubated with a hydroxamic acid inhibitor, 0.01 – 0.3 µM or

LTA4, 1-10 µM. Aminopeptidase assays were then performed as described.

Crystallization

Plate-like crystals of [R563A]LTA4H were obtained by liquid-liquid diffusion in capillaries,

as previously described (26). Briefly, 5 µl of precipitation solution (28 % (v/v) PEG8000, 0.1

mM Na acetate, 0.1 mM imidazole buffer, pH 6.8, and 5 mM YbCl3), was injected into the

bottom of a melting point capillary and an equal volume of [R563A]LTA4H (5 mg/ml) in 10

mM Tris-HCl, pH 8, containing 1 mM bestatin, was layered on top.

Data collection and structure determination

For data collection, crystals were soaked in 15% (w/v) PEG8000, 50 mM sodium-acetate, 50

mM imidazole buffer, pH 6.8, 2.5 mM YbCl3 and 25% (v/v) glycerol. Data was collected at

the beamline I711 of Max-Lab, Lund, Sweden. A complete set of data was collected from a

single crystal. Statistics on data collection and quality are given in Table I. The crystals

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belonged to space-group P212121 with cell dimensions a=78 b=86.85 c=98.95, α=β=γ=90° at

100 K. Processing, scaling and merging of data were carried out using the program Mosflm

(33) and programs from CCP4 (34). The [E271Q]LTA4H mutant structure (25) was used as

the starting point for refinement. All refinement was done using the CNS package (35).

Manual model building as well as interpretation of electron density maps was performed

using the program XtalView (36). During the refinement, 614 water molecules, one Zn2+ ion,

one imidazole molecule, one acetate molecule, and one Yb3+ ion were identified. The final R-

factor was 18.8 % and the Rfree factor was 23.9 %. 3.3% of total reflections were set aside to

calculate the Rfree. Most of the model of [R563A]LTA4H is in good density except for the

(His)6-tag and the first four N-terminal residues. In the model, 99.4 % of the residues are

confined to the most favorable or additionally allowed regions and 0.6 % in the generously

allowed regions of the Ramachandran plot. R.m.s. deviations for bond lengths and angles

were 0.0079 Å and 1.44°.

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Results

Mutagenetic replacements, expression and purification of recombinant proteins

To detail the function of the Arg/Lys site in LTA4H we exchanged Arg563 for a Lys, Ala or

Met, and Lys565 for an Arg, Ala or Met, generating the mutants [R563K]-, [R563A]-,

[R563M]-, [K565R]-, [K565A]- and [K565M]LTA4H. We also reversed the positions of

Arg563 and Lys565, generating the double cross-over mutant [R563K/K565R]LTA4H. As

control, we exchanged Glu560 for a Gln, generating [E560Q]LTA4H. This control residue

was selected on the basis of proximity to the active site and predicted non-catalytic nature.

The resulting 8 mutants were all expressed as (His)6-tagged fusion proteins in E. coli, to allow

rapid purification on nickel-NTA resins. The level of expression was similar for wild type

enzyme and all mutants, with a final yield of 2.4 to 6.5 mg purified protein per liter cell

culture.

Catalytic properties of mutants of Arg563

Mutagenetic replacements of Arg563 completely eliminated the epoxide hydrolase activity of

LTA4H (Table II). Thus, neither [R563K]-, [R563A]-, nor [R563M]LTA4H converted LTA4

into detectable amounts of LTB4. Although these mutations also strongly reduced the

aminopeptidase activity, the effects were not quite as detrimental as for the epoxide hydrolase

reaction. Thus, in [R563K]LTA4H, Vmax and Km reached 6.4 and 58%, respectively, of wild

type enzyme, while in [R563A]LTA4H, the aminopeptidase activity was almost completely

abolished, with a Vmax and Km for Ala-p-NA of 0.3 and 6.2 %, respectively. On the other

hand, [R563M]LTA4H exhibited a Vmax and Km of 20 and 179%, respectively, of wild type

enzyme (Table II). Accordingly, the specificity constants of both [R563K]- and

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[R563M]LTA4H were reduced to only 11% of the wild type enzyme, whereas the specificity

constant for [R563A]LTA4H only reached 5%.

Catalytic properties of mutants of Lys565

Mutations of Lys565 had variable, but no dramatic effects on the epoxide hydrolase activity.

[K565R]LTA4H exhibited a Vmax and Km of 95 and 104%, respectively, of wild type enzyme,

whereas for [K565A]LTA4H, Vmax and Km for LTA4 were 31 and 14%, respectively, and for

[K565M]LTA4H these values mounted to 32 and 33%, respectively, of wild type enzyme

(Table II). In addition, the specificity constants for the more conservative mutant

[K565R]LTA4H and for [K565M]LTA4H remained unchanged (95 and 92%, respectively)

and even increased for [K565A]LTA4H (218%), indicating that the substrate binding and

catalytic residues are not drastically altered in these mutants. For the aminopeptidase activity,

on the other hand, exchange of Lys565 for an Ala or Met mostly affected the Michaelis

constant, with only marginal effects on the Vmax value, whereas exchange of Lys565 for an

Arg affected neither Vmax nor Km. Thus, for [K565A]LTA4H, Vmax and Km were 78 and 483%,

respectively, of the wild type enzyme and for [K565M]LTA4H the corresponding values were

58 and 645%, respectively. For [K565R]LTA4H, which retains the positive charge, values of

Vmax and Km remained at 108 and 82%, respectively (Table II). The effects on the Michaelis

constants were also reflected in the specificity constants of [K565A]- and [K565M]LTA4H,

which were reduced to 16 and 9%, respectively, but increased minimally in [K565R]LTA4H

to 132%. It should be noted that Lys565 is part of a salt bridge involving Glu533 and that

effects of mutations may, at least to some extent, be explained by a disturbance of these

charge interactions.

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Control mutants [R563K/K565R]- and [E560Q]LTA4H

As expected, the double cross-over mutant [R563K/K565R]LTA4H, yielded an enzyme

devoid of epoxide hydrolase activity, as seen in [R563K]LTA4H. However,

[R563K/K565R]LTA4H exhibited a reduced peptidase activity, exhibiting a Vmax and Km of

18 and 47%, respectively, of wild type enzyme. The specificity constant for the double cross-

over mutant attained 39% of that of wild type enzyme.

The control mutation close to Arg563 and Lys565 did not cause any major effects on

enzyme catalysis. For the epoxide hydrolase activity, [E560Q]LTA4H exhibited a Vmax and

Km of 61 and 60%, respectively, of wild type enzyme (Table II). As a consequence, the

specificity constant was not significantly changed. For the aminopeptidase activity,

[E560Q]LTA4H exhibited a Vmax and Km of 39 and 109%, respectively, of wild type enzyme

(Table II). The kcat/Km value for [E560Q]LTA4H was reduced to 36%.

Analysis of LTA4 binding strength by enzyme inhibition assays

Since all mutants in position 563 lacked epoxide hydrolase activity, inhibition assays against

the residual aminopeptidase activity were carried out to probe the effects of mutations on the

binding of LTA4. For [R563K]- and [R563M]LTA4H, the hydroxamic acid inhibitor

exhibited a Ki of 0.1 and 0.2 µM, as compared to 0.01 µM for wild type enzyme. In contrast,

no significant difference in Ki could be detected between [R563K]-, [R563M]- and wild type

LTA4H when employing LTA4 as an inhibitor of the aminopeptidase activity (Table III).

Thus, the Ki for LTA4 (0-10 µM) was determined to 7.5, 5.5 and 8 µM for [R563K]-,

[R563M]- and wild type LTA4H, respectively. These values are similar to previously reported

Km values for LTA4 (5-30 µM), suggesting that the substrate binds equally strong and in

similar conformations to both mutated and wildtype LTA4H.

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Crystal structure of [R563A]LTA4H

The overall structure of [R563A]LTA4H is very similar to that of wild type LTA4H (26),

with an overall r.m.s. difference of all Cα atoms of 0.39 Å, and for all protein atoms 0.7 Å

between the two structures. The structure of [E271Q]LTA4H (25) was used to calculate initial

electron density maps. In the initial Fobs-Fcalc electron density map, negative densities were

observed around the side-chain of residue 563, verifying the correct exchange of the original

Arg for an Ala. In the finally refined structure of [R563A]LTA4H, only the loop between

residues 175 and 185 differs from its conformation in the wild type enzyme, the reason for

which could be differences in crystal packing between the two different space groups. Both

wild type LTA4H and [R563A]LTA4H were co-crystallized with bestatin. Whereas in the

Fobs-Fcalc electron density map of wild type LTA4H bestatin was readily identified, no

features interpretable as this tight-binding inhibitor were found in the Fobs-Fcalc electron

density map of [R563A]LTA4H. The local environment of the region of mutation exhibits a

slight structural modification, in which the loss of the arginyl side-chain gives rise to an

increased, locally available, space which displaces the Nζ of Lys565 about 2.5 Å towards the

initial position of Arg563 (Fig. 2).

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Discussion

The biological effects of LTB4 are signaled via the BLT1 receptor present on the surface of

immuno-competent cells, e.g. peripheral leukocytes, including neutrophils and eosinophils, as

well as peritoneal macrophages (37,38). The chemical properties of LTB4 that are crucial for

agonistic action at the BLT1 receptor include a ∆6−cis-∆8-trans-∆10-trans double bond

geometry and an R-configuration of the hydroxyl group at C12, as determined by structure-

activity studies (21-23). Previous work on mutated enzymes and non-mammalian LTA4H

have indicated that a precise alignment of LTA4 in the active site is also very important for

catalysis and formation of the proper chemistry in the product LTB4 (18,39-41). This

conclusion is further corroborated by the crystal structure of LTA4H, which suggests that the

critical ∆6-cis double bond in LTB4 may be governed by the specific alignment of LTA4 along

an L-shaped, putative substrate-binding tunnel (26). Apparently, recognition of the substrate

LTA4 precedes these events and must be a key factor for successful catalysis.

In the X-ray crystal structure of LTA4H, two positively charged residues, Arg563 and

Lys565, are located in a wide portion of the putative substrate binding cavity, near its

entrance, and in the vicinity of the carboxyl group of bestatin. To explore the potential

involvement of Arg563 and Lys565 in the recognition, binding and turnover of substrates, we

carried out a combined mutational and structural analysis of these residues.

Arg563 is critical for turnover of LTA4

Mutations of Arg563 resulted in complete loss of catalytic function. Surprisingly, the

conservative replacement of Arg for Lys, which preserves the positive charge, also abolished

enzyme function, indicating that Arg563 plays a critical role in substrate turnover (Table II).

This conclusion is supported by the structure of [R563A]LTA4H which did not reveal any

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structural alterations that could explain the complete loss of catalytic activity (Fig. 2). In

addition, the mutant [R563K]LTA4H exhibited a significant residual aminopeptidase activity

indicating that no major structural changes had occurred at the active site.

Mutations of Arg563 do not compromise binding strength for LTA4

Since [R563K]LTA4H did not display any epoxide hydrolase activity, values of Km could not

be determined as a measure of LTA4 binding. Instead, we used an indirect approach, taking

advantage of the residual peptidase activity of [R563K]- and [R563M]LTA4H, to evaluate the

contribution of Arg563 to the binding strength between LTA4 and the protein. Thus, we

analyzed the effects of mutations on the inhibitory efficiency of LTA4 itself and a tight-

binding hydroxamic acid inhibitor that was designed as a mimic of LTA4 (42). The structure

of LTA4H complexed with this inhibitor has been determined (28). In the structure, the

inhibitor occupies almost entirely the proposed LTA4 binding site, and its carboxyl end makes

a direct salt bridge with Arg563. For [R563K]- and [R563M]LTA4H, the Ki values for LTA4

were 7.5 and 5.5 µM, respectively, as compared to 8 µM for the wild type enzyme, indicating

that exchange of Arg for Lys or Met did not affect substrate binding strength. This finding

may be interpreted in two ways, either Arg563 does not contribute that much to the overall

binding strength between LTA4 and the protein, and/or the carboxylate tail of LTA4 binds in

alternative conformations, with similar strength, in the mutated enzymes. The corresponding

Ki values for the hydroxamic acid were 0.1, 0.2 and 0.01 µM for [R563K]-, [R563M] and

wild type LTA4H, respectively. The reduced efficiency of the hydroxamic acid in

[R563K]LTA4H most probably reflects an increased space between the zinc and the positive

charge of Lys, which in turn may affect zinc-hydroxamate interactions. In addition, an Arg

side-chain provides two bonds for carboxylate interaction, which increases the precision of

binding, whereas a Lys only provides a single bond (43). Removing the charge by

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replacement of Arg for Met only caused a marginal additional reduction of inhibitor potency.

Taken together, these data demonstrate that the loss of epoxide hydrolase activity in

[R563K]LTA4H is not related to a decreased binding strength between substrate and protein

and further corroborate the notion that Arg563, in an indirect manner (see below), is required

for successful turnover of LTA4 into LTB4.

Arg563 is an atypical recognition site for the carboxyl group of LTA4

The original proposal that arginyl residues are common anion recognition sites in many, if not

all, enzymes utilizing anionic substrates is now a well-established fact in enzymology (44). It

holds true also for enzymes involved in fatty acid metabolism, e.g. cytochrome p450BM-3

(45) and central enzymes in the eicosanoid cascade such as COX-1 and COX-2 (46-50). The

usual role of the positively charged residue is to guide the incoming substrate and provide

firm anchoring to the protein. Thus, mutations of carboxylate recognition sites normally lead

to a reduced activity due to increased Km for the substrate. In this respect, LTA4H appears to

be an unusual example of an enzyme that is critically dependent on the Arg residue for

enzyme function, rather than achieving sufficient substrate binding strength.

Arg563 ensures precision in LTA4 alignment at the active site

It has been known since long that esterified LTA4 is not a substrate for LTA4H (51), which

has sometimes been interpreted as the result of steric hindrance at the active site. This strong

specificity for the free acid of LTA4 has also been taken as evidence for a catalytic

mechanism involving a substrate-catalyzed, carboxylic acid-induced, activation of the epoxide

moiety as an initial step in the enzymatic reaction (29). From the present data, however, it

seems clear that the absolute requirement of a free C1 carboxylate is best explained by the

need of a precise positioning of the substrate, via Arg563, to guarantee optimal alignment

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necessary for catalysis (Fig. 3A). The neighboring residue, Lys565, which is also located such

that it could well be involved in carboxylate recognition, seems to have little, if any, role in

the binding and turnover of LTA4 (Table II). Thus, the epoxide hydrolase activities of

[K565R]-, [K565A]- and [K565M]LTA4H were not significantly decreased.

The C-terminal anchoring of peptide substrates appear to involve both Arg563 and Lys565

Mutations of Arg563 reduced the aminopeptidase activity of LTA4H albeit to a variable

degree. The activity of [R563A]LTA4H against Ala-p-NA was barely detectable, whereas

[R563K]- and [R563M]LTA4H were catalytically less compromised with a significant

residual activity of 6.4 and 20%, respectively, as compared to the wild type enzyme (Table

II). These effects suggest that Arg563 is a carboxylate binding site also for peptide substrates

(Fig. 3B). However, unlike the anchoring of LTA4 in the epoxide hydrolase reaction, Arg563

seems to be assisted by Lys565 in the recognition of peptides. Thus, when compared to wild

type enzyme, the turnover of Ala-p-NA by [K565A]-, [K565M]- and [K565R]LTA4H was

only slightly affected whereas a distinct increase in the Michaelis constant was observed for

[K565A]- and [K565M]LTA4H, i.e. the two mutants lacking positive charge (Table II).

The natural peptidase substrate(s) of LTA4H is currently not known but the enzyme can

hydrolyze a variety of compounds containing a peptide or amide bond. One of the best and

most frequently used substrates is Ala-p-NA, which is used as a model substrate for the

aminopeptidase reaction. Although molecular docking experiments indicate that Ala-p-NA

aligns in the catalytic pocket with the nitro-group in a similar orientation to that of the

carboxylate of tripeptide substrates (25), these two functional groups are not equivalent. The

carboxylate has a negative net charge, whereas the nitro-group, which exhibits a delocalized

charge, has a net charge of zero. Therefore, Ala-p-NA is not a perfect aminopeptidase

substrate, which limits our possibilities to predict the behavior of natural peptide substrate(s).

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However, crystallographic data offer further support for our conclusions. In the crystal

structure of wild type LTA4H, the tight-binding, competitive, inhibitor bestatin was included.

Bestatin is also a dipeptide mimetic and in the structure, the carboxylate moiety of the

molecule appears to interact with Arg563 and Lys565 via an intervening water molecule,

suggesting that the C-terminus of a dipeptide substrate may bind in a similar manner (26).

Also, in the crystal structure of LTA4H complexed with captopril, a direct interaction was

seen between this inhibitor and Arg563 (28). Bestatin was included in the crystallization of

[R563A]LTA4H, but in the 3D-structure the compound could not be identified at the active

site, suggesting absence or low occupancy of the inhibitor, possibly due to loss of binding to

Arg563. Taken together, the mutagenetic and crystallographic data indicate that Arg563 is

responsible for the basal binding of the C-terminus of a peptide substrate required for an

alignment compatible with catalysis. Lys565, on the other hand, seems to contribute

significant binding strength and thus cooperates with Arg563 to achieve optimal conditions

for efficient substrate turnover (Fig. 3B).

The carboxylate recognition site, Arg563/Lys565, is a unique feature of LTA4H

LTA4H belongs to the M1 family of zinc metallopeptidases, which is a subfamily of the

broader MA clan of “thermolysin-like” metalloproteases (52). A common structural feature in

this large group of enzymes is the zinc binding motif HEXXH-(X)18 -E (53) and in LTA4H,

the catalytic zinc is coordinated by His295, His299 and Glu318 (54). More specifically, the

M1 family includes enzymes such as aminopeptidase A (EC 3.4.11.7, APA), aminopeptidase

B (EC 3.4.11.6, APB) and aminopeptidase N (EC 3.4.11.2, APN), all of which share a

conserved GXMEN motif. This signature harbors a Glu residue acting as an N-terminal

recognition site for peptide substrates. In LTA4H, the corresponding Glu271 is also critical

for the epoxide hydrolase reaction, presumably in the early activation and opening of the

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epoxide moiety (25). In the tertiary structure, most catalytic residues of LTA4H, e.g. Glu271,

Glu296, Asp375 and Tyr383 are located within the central catalytic domain. This domain is

structurally very similar to thermolysin and is possibly a common structural feature for the

entire MA clan of metalloproteases, which include the M1 metallopeptidases (55). However,

unlike the other catalytic residues, Arg563 and Lys565 are located within the C-terminal

domain, which seems to be the segment of the protein that is most typical for LTA4H (26).

Considering the role of Glu271 as an anchor for the α-amino group of a peptide substrate

(25), the Arg/Lys site may limit the length of the substrate and thus becomes an important

determinant for the tripeptide specificity of LTA4H. Concerning the evolutionary

preservation of Arg563 and Lys565, these residues are highly conserved among LTA4H

homologues across a wide range of species, but show less conservation between other

members of the M1 family (Fig. 4). However, only one of the non-mammalian homologues,

viz. S. cerevisiae LTA4H, has been shown to possess an epoxide hydrolase activity (41) and

the enzyme from C. elegans has even been reported to lack this activity (56). In other

mammalian M1 metallopeptidases such as APA and APN, this site is not conserved since the

domain in which these residues are found is replaced by another domain sequence.

Among other M1 metallopeptidases, APB seems to exhibit the highest degree of

identity with LTA4H over the C-terminal domain and also carries a Lys/Lys site, suggesting

that APB may well be a tripeptidase. This hypothesis agrees well with earlier work with

human and rat APB, showing that di- and tripeptides are good substrates whereas

oligopeptides are not hydrolyzed (57). In any event, our data suggest that evolution has

developed Arg563 and Lys565 to carry out a fundamental function in carboxylate recognition

as well as catalytic turnover of both lipid and peptide substrates, in the common active center

of LTA4H.

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Acknowledgements.

We thank personnel at beamline I711 of MAX-Lab, Lund for help during data collection and

Eva Ohlson for technical assistance. The work was funded by the Swedish Medical Research

Council (O3X-10350), The European Union (QLG1-CT-2001-01521), The Swedish

Foundation for Strategic Research (fellowships to P.C.R. & M.A.), The Wallström

Foundation, Konung Gustav V:s 80-Årsfond, the Swedish Natural Sciences Research Council

(M.T.), and AFA Health Insurances.

Coordinates

Coordinates of [R563A]LTA4H have been deposited in the Protein Data Bank, accession

code 1SQM.

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References

1. Samuelsson, B., Dahlén, S.-E., Lindgren, J.-Å., Rouzer, C. A., and Serhan, C. N. (1987) Science 237, 1171-1176

2. Funk, C. D. (2001) Science 294, 1871-1875 3. Samuelsson, B. (1983) Science 220, 568-575 4. Payan, D. G., Missirian-Bastian, A., and Goetzl, E. J. (1984) Proc. Natl. Acad. Sci.

USA 81, 3501-3505 5. Yamaoka, K. A., Claesson, H.-E., and Rosén, A. (1989) J. Immunol. 143, 1996-2000 6. Tager, A. M., Bromley, S. K., Medoff, B. D., Islam, S. A., Bercury, S. D., Friedrich,

E. B., Carafone, A. D., Gerszten, R. E., and Luster, A. D. (2003) Nat. Immunol. 4, 982-990

7. Ott, V. L., Cambier, J. C., Kappler, J., Marrack, P., and Swanson, B. J. (2003) Nat. Immunol. 4, 974-981

8. Goodarzi, K., Goodarzi, M., Tager, A. M., Luster, A. D., and von Andrian, U. H. (2003) Nat. Immunol. 4, 965-973

9. Bailie, M. B., Standiford, T. J., Laichalk, L. L., Coffey, M. J., Strieter, R., and Peters-Golden, M. (1996) J. Immunol. 157, 5221-5224

10. Mancuso, P., Nana-Sinkam, P., and Peters-Golden, M. (2001) Infect. Immun. 69, 2011-2016

11. Chen, X. S., Sheller, J. R., Johnson, E. N., and Funk, C. D. (1994) Nature 372, 179-182

12. Byrum, R. S., Goulet, J. L., Griffiths, R. J., and Koller, B. H. (1997) J. Exp. Med. 185, 1065-1075

13. Byrum, R. S., Goulet, J. L., Snouwaert, J. N., Griffiths, R. J., and Koller, B. H. (1999) J. Immunol. 163, 6810-6819

14. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y., and Shimizu, T. (1997) Nature 387, 620-624

15. Yokomizo, T., Kato, K., Terawaki, K., Izumi, T., and Shimizu, T. (2000) J. Exp. Med. 192, 421-431

16. Haeggström, J. Z. (2000) Am. J. Resp. Crit. Care Med. 161, 25-31 17. Mueller, M. J., Wetterholm, A., Blomster, M., Jörnvall, H., Samuelsson, B., and

Haeggström, J. Z. (1995) Proc. Natl. Acad. Sci. USA 92, 8383-8387 18. Mueller, M. J., Blomster, M., Oppermann, U. C. T., Jörnvall, H., Samuelsson, B., and

Haeggström, J. Z. (1996) Proc. Natl. Acad. Sci. USA 93, 5931-5935 19. Blomster Andberg, M., Hamberg, M., and Haeggström, J. Z. (1997) J. Biol. Chem.

272, 23057-23063 20. Rudberg, P. C., Tholander, F., Thunnissen, M. M., Samuelsson, B., and Haeggström,

J. Z. (2002) Proc. Natl. Acad. Sci. USA 99, 4215-4220 21. Ford-Hutchinson, A. W., Bray, M. A., Cunningham, F. M., Davidson, E. M., and

Smith, M. J. (1981) Prostaglandins 21, 143-152 22. Leblanc, Y., Fitzsimmons, B. J., Charleson, S., Alexander, P., Evans, J. F., and

Rokach, J. (1987) Prostaglandins 33, 617-625 23. Yokomizo, T., Kato, K., Hagiya, H., Izumi, T., and Shimizu, T. (2001) J. Biol. Chem.

276, 12454-12459 24. Örning, L., Gierse, J. K., and Fitzpatrick, F. A. (1994) J. Biol. Chem. 269, 11269-

11273 25. Rudberg, P. C., Tholander, F., Thunnissen, M. M., and Haeggström, J. Z. (2002) J.

Biol. Chem. 277, 1398-1404 26. Thunnissen, M. G. M., Nordlund, P., and Haeggström, J. Z. (2001) Nat. Struct. Biol. 8,

by guest on April 8, 2018

http://ww

w.jbc.org/

Dow

nloaded from

23

131-135 27. Mueller, M. J., Samuelsson, B., and Haeggström, J. Z. (1995) Biochemistry 34, 3536-

3543 28. Thunnissen, M. M., Andersson, B., Samuelsson, B., Wong, C. H., and Haeggström, J.

Z. (2002) FASEB J. 16, 1648-1650 29. Ollmann, I. R., Hogg, J. H., Munoz, B., Haeggström, J. Z., Samuelsson, B., and

Wong, C.-H. (1995) Bioorg. Med. Chem. 3, 969-995 30. Sarkar, G., and Sommer, S. S. (1990) Biotechniques 8, 404-407 31. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 32. Wetterholm, A., Haeggström, J. Z., Samuelsson, B., Yuan, W., Munoz, B., and Wong,

C.-H. (1995) J. Pharmac. Exp. Ther. 275, 31-37 33. Leslie, A. G. W. (1992) Joint CCP4 and ESF-EACMB Newsletter on Protein Crystallography 34. Collaborative Computing Project Number 4. (1994) Acta Crystallogr. D 50, 760-763 35. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-

Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. D 54, 905-921

36. McRee, D. E. (1999) J. Struct. Biol. 125, 156-165 37. Huang, W. W., Garcia-Zepeda, E. A., Sauty, A., Oettgen, H. C., Rothenberg, M. E.,

and Luster, A. D. (1998) J. Exp. Med. 188, 1063-1074 38. Yokomizo, T., Izumi, T., and Shimizu, T. (2001) Arch. Biochem. Biophys. 385, 231-

241 39. Mueller, M. J., Andberg, M. B., Samuelsson, B., and Haeggström, J. Z. (1996) J. Biol.

Chem. 271, 24345-24348 40. Kull, F., Ohlson, E., and Haeggström, J. Z. (1999) J. Biol. Chem. 274, 34683-34690 41. Kull, F., Ohlson, E., Lind, B., and Haeggström, J. Z. (2001) Biochemistry 40, 12695-

12703 42. Hogg, J. H., Ollmann, I. R., Wetterholm, A., Blomster Andberg, M., Haeggström, J.,

Samuelsson, B., and Wong, C.-H. (1998) Chem. Eur. J. 4, 1697-1713 43. Witt, S., Wohlfahrt, G., Schomburg, D., Hecht, H. J., and Kalisz, H. M. (2000)

Biochem. J. 347, 553-559 44. Riordan, J. F., McElvany, K. D., and Borders, C. L., Jr. (1977) Science 195, 884-886 45. Li, H., and Poulos, T. L. (1997) Nat. Struct. Biol. 4, 140-146 46. Picot, D., Loll, P. J., and Garavito, R. M. (1994) Nature 367, 243-249 47. Mancini, J. A., Riendeau, D., Falgueyret, J. P., Vickers, P. J., and O'Neill, G. P. (1995)

J. Biol. Chem. 270, 29372-29377 48. Bhattacharyya, D. K., Lecomte, M., Rieke, C. J., Garavito, M., and Smith, W. L.

(1996) J. Biol. Chem. 271, 2179-2184 49. Luong, C., Miller, A., Barnett, J., Chow, J., Ramesha, C., and Browner, M. F. (1996)

Nat. Struct. Biol. 3, 927-933 50. Rieke, C. J., Mulichak, A. M., Garavito, R. M., and Smith, W. L. (1999) J. Biol.

Chem. 274, 17109-17114 51. Maycock, A. L., Anderson, M. S., DeSousa, D. M., and Kuehl, F. A., Jr. (1982) J.

Biol. Chem. 257, 13911-13914 52. Barret, A. J., Rawlings, N. D., and Woessner, J. F. (1998) in Handbook of proteolytic

enzymes (Barret, A. J., Rawlings, N. D., and Woessner, J. F., eds), pp. 992-993, Academic Press, London, San Diego

53. Vallee, B. L., and Auld, D. S. (1990) Biochemistry 29, 5647-5659 54. Medina, J. F., Wetterholm, A., Rådmark, O., Shapiro, R., Haeggström, J. Z., Vallee,

B. L., and Samuelsson, B. (1991) Proc. Natl. Acad. Sci. USA 88, 7620-7624

by guest on April 8, 2018

http://ww

w.jbc.org/

Dow

nloaded from

24

55. Barret, A. J., Rawlings, N. D., and Woessner, J. F. (1998) in Handbook of proteolytic enzymes (Barret, A. J., Rawlings, N. D., and Woessner, J. F., eds), pp. 994-996, Academic Press, London, San Diego

56. Baset, H. A., Ford-Hutchinson, A. W., and O'Neill, G. P. (1998) J. Biol. Chem. 273, 27978-27987

57. Söderling, E. (1983) Arch. Biochem. Biophys. 220, 1-10 58. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723 59. Kraulis, P. J. (1991) J. Appl. Cryst. 24, 946-950 60. Wetterholm, A., Medina, J. F., Radmark, O., Shapiro, R., Haeggström, J. Z., Vallee,

B. L., and Samuelsson, B. (1992) Proc. Natl. Acad. Sci. USA 89, 9141-9145 61. Blomster, M., Wetterholm, A., Mueller, M. J., and Haeggström, J. Z. (1995) Eur. J.

Biochem. 231, 528-534

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Figure legends

FIG. 1. Stereo view of LTA4H, spatial relationships between residues and the substrate

LTA4 at the catalytic zinc site

In this stereo image, wild type LTA4H is depicted in a ball- and stick model. LTA4 was

modeled into the putative hydrophobic, L-shaped, catalytic pocket. The carboxyl group of

LTA4 faces the side-chains of Arg563 and Lys565. The catalytic residues Glu271 (25) and

Asp375 (20) are shown, and the catalytic zinc is depicted as a silver sphere. The figure was

generated with Swiss-PdbViewer (58) and POV-Ray (http://www.povray.org).

FIG. 2. Stereo view of the loop 562 - 566 of [R563A]LTA4H in Fobs-Fcalc electron density

For the calculation of Fcalc and αcalc, the atoms of loop 562-566 were excluded from the

calculations. The density was contoured at 3σ level. The picture was made using Molscript

(59), Glr (L. Esser & J. Deisenhofer, personal communications) and POV-Ray

(http://www.povray.org).

FIG. 3. Models for the catalytic mechanisms in LTA4H

(A) In the epoxide hydrolase reaction a water molecule is polarized by Glu271 and the

catalytic zinc (25), to promote an acid-induced opening of the epoxide. This reaction yields a

carbocation intermediate and finally a nucleophilic attack, guided by Asp375, occurs at C12

(20). In this reaction Arg563 serves in carboxylate recognition and substrate alignment.

Dotted lines indicate interactions between important groups. (B) In the aminopeptidase

reaction, Glu271 is involved in N-terminal recognition (25), Glu296 and the catalytic zinc act

as base catalyst and Tyr383 functions as proton donor (60,61). Here, Arg563 and Lys565

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serve together in carboxylate recognition. Dotted lines indicate interactions between

important groups. See text for further details.

FIG. 4. Sequence alignment of members of the M1 family of the MA clan of

metallopeptidases

The figure shows residues subjected to mutational analysis, as well as the three zinc ligands in

members of the M1 family of metallopeptidases. Note the strict conservation of the zinc

ligands (His295, His299 and Glu318), as opposed to the carboxylate recognition site. The

sequence alignment was created using CLUSTALW at Biology WorkBench 3.2

(http://workbench.sdsc.edu).

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TABLE I. Data collection and refinement statistics.

Data collection statistics

Diffraction limit (Å) 2.3

Wavelength (Å) 0.968

Completeness (%) 99.7 (99.7)

Mean I/σ (I) 5.3 (3)

Multiplicity of observation 4.7 (4.7)

Rmergea (%) 9.2 (23.1)

Refinement statistics

R-factorb (%) 18. 8

Rfreec (%) 23.9

R.m.s.d. in bond distance (Å) 0.0079

R.m.s.d. in bond angle (°) 1.44

a Rmerge = ΣhΣi|Ii (h) - I(h)|/ ΣhΣiIi (h), where Ii (h) is the ith measurement of reflection h and

I(h) is the weighted mean of all measurements of h.

b R-factor = Σh|Fobs (h) – Fcalc (h)|/ ΣhFobs (h), where

Fobs and Fcalc are the observed and calculated factor amplitudes, respectively.

c Rfree is the R-factor calculated for the test set of reflections (3.3 %) which are

omitted during the refinement process.

Data for the highest resolution shell (2.3–2.42 Å) are shown within parantheses.

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TABLE II. Kinetic parameters for the aminopeptidase and epoxide hydrolase activities

Mutated recombinant enzymes were expressed in E. coli, purified by affinity chromatography

and assayed for the aminopeptidase and epoxide hydrolase activity. Apparent kinetic

constants are expressed in % of those of wild type enzyme. Each data point was calculated as

mean, ± s.e., triplicate determinations. n.d. = not detectable.

Epoxide hydrolase activity

(% of WT) Aminopeptidase activity

(% of WT)

Mutant Vmax Km kcat/Km Vmax Km kcat/Km

Wild type 100 ± 7 100 ± 25 100 100 ± 2 100 ± 5 100

E560Q 61 ± 3 60 ± 12 100 39 ± 4 109 ± 10 36

R563K n.d. n.d. 6.4 ± 0.9 58 ± 15 11

R563A n.d. n.d. 0.3 ± 0.01 6.2 ± 1.6 5

R563M n.d. n.d. 20 ± 2 179 ± 37 11

K565R 95 ± 12 104 ± 33 92 108 ± 17 82 ± 21 132

K565A 31 ± 4 14 ± 16 218 78 ± 12 483 ± 110 16

K565M 32 ± 2 33 ± 12 95 58 ± 12 645 ± 173 9

R563K/K565R n.d. n.d. 18 ± 3 47 ± 14 39

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TABLE III. Inhibition assays

Ki (µM)

Inhibitor WT R563K R563M

Hydroxamic acid 0.01 0.1 0.2

LTA4 8 7.5 5.5

Values for inhibition constants (Ki) are in µM and were determined by Dixon plots. The

hydroxamic acid-like inhibitor was used in concentrations between 0.01 and 0.3 µM. LTA4

was used in concentrations between 0 and 10 µM.

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and Jesper Z. HaeggströmPeter C. Rudberg, Fredrik Tholander, Martina Andberg, Marjolein M.G.M. Thunnissen

for the epoxide hydrolase and aminopeptidase substratesLeukotriene A4 hydrolase: Identification of a common carboxylate recognition site

published online April 12, 2004J. Biol. Chem. 

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