Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, 171 77 Stockholm, SWEDEN

LEUKOTRIENE A4 HYDROLASE

Identification of amino acid residues involved in catalyses and substrate-mediated inactivation

Martin J. Mueller

Stockholm 2001

Published and printed by Karolinska University Press Box 200, SE-171 77 Stockholm, Sweden © Martin J. Mueller, 2001 ISBN 91-628-4934-4

Abstract

Leukotriene (LT) A4 hydrolase catalyzes the committed step in the biosynthesis of LTB4, a classical chemoattractant and immune-modulating lipid mediator involved in , host-defense against infections, and systemic, PAF-mediated, lethal shock. LTA4 hydrolase is a bifunctional zinc metalloenzyme with a chloride-stimulated arginyl aminopeptidase activity. When exposed to its lipid substrate LTA4, the is inactivated and covalently modified in a process termed suicide inactivation, which puts a restrain on the enzyme's ability to form the biologically active LTB4. In the present thesis, chemical modification with a series of amino acid-specific reagents, in the presence and absence of competitive inhibitors, was used to identify catalytically important residues at the active site. Thus, using differential labeling techniques, modification with the tyrosyl reagents N-acetylimidazole and tetranitromethane revealed the presence of two catalytically important Tyr residues. Likewise, modification with 2,3-butanedione and phenylglyoxal indicated that three Arg residues were located at, or near, the active center of the enzyme. Using differential Lys-specific peptide mapping of untreated and suicide inactivated LTA4 hy- drolase, a 21 residue peptide termed K21, was identified that is involved in binding of LTA4 to the native protein. Isolation and amino acid sequencing of a modified form of K21, revealed that Tyr- 378 is the site of attachment between LTA4 and the protein. To investigate the functional role of Tyr-378, this residue was subjected to mutational analysis. Thus, Tyr-378 was exchanged for a Phe and Gln residue and the purified recombinant were characterized. Upon exposure to LTA4, none of the mutated enzymes were susceptible to inactivation and covalent modification by LTA4. In addition, the most conservative mutation generated an enzyme, [Y378F]LTA4 hydrolase, that exhibited an increased (2.5 fold) turnover of LTA4 into LTB4, presumably due to a reduced catalytic restrain normally imposed by suicide inactivation. Hence, by a single point mutation we generated an enzyme that is protected from suicide inactivation and exhibits an increased catalytic efficiency. Moreover, we had shown that catalysis and covalent modification/inactivation could be completely dissociated. Both mutants of Tyr-378 were able to convert LTA4 into a novel product that was structurally identified by UV spectroscopy, GC/MS, UV-induced double-bond isomerization, and comparisons with synthetic standards. Thus, we could show that these two mutants could generate both LTB4 6 8 and the double-bond isomer ∆ -trans-∆ -cis-LTB4. The fact that mutation of Tyr-378 allows forma- tion of a double-bond isomer of the natural product LTB4, suggests that this residue may be in- volved in the alignment of the substrate, or a carbocation derived thereof, in the active site. To detail the molecular mechanism for substrate-mediated inactivation of LTA4, further analysis of mutated enzymes with peptide mapping and enzyme activity determinations was carried out. Thus, we exposed the functionally incompetent mutant [E318A]LTA4 hydrolase for the substrate LTA4, and found that this protein was covalently modified to the same, or even higher, extent as the catalytically active wild type enzyme. Hence, the catalytic machinery is not required to activate the substrate to a molecular species that can bind to the protein. Together with other data from our labo- ratory as well as the inherent chemical properties of LTA4, we conclude that substrate-mediated inactivation of LTA4 hydrolase follows an affinity labeling mechanism, rather than a mechanism- based process. This conclusion predicts (i) that LTA4 is likely to destroy other enzymes/protein in its neighborhood to which it binds with sufficient strength and (ii) that LTA4 can not be used as a template for design of a suicide-type inhibitors.

ISBN 91-628-4934-4

This thesis is based on the following articles, which will be referred to in the text by their Roman numerals.

I. Mueller, M.J., Samuelsson, B and Haeggström, J.Z. (1995) Chemical modification of hydrolase. Indications for essential tyrosyl and arginyl residues at the active site. Biochemistry 34, 3536-3543.

II. Mueller, M.J., Wetterholm, A., Blomster, M., Jörnvall, H., Samuelsson, B. and Haeggström, J.Z. (1995) Leukotriene A4 hydrolase: Mapping of a henicosapeptide involved in mechanism- based inactivation. Proc. Natl. Acad. Sci. USA 92, 8383-8387.

III. Mueller, M.J., Blomster, M., Oppermann, U.C.T., Jörnvall, H., Samuelsson, B. and Haeggström, J.Z. (1996) Leukotriene A4 hydrolase: Protection from mechanism-based inactivation by mutation of tyrosine-378. Proc. Natl. Acad. Sci. USA 93, 5931-5935.

IV. Mueller, M.J., Blomster Andberg, M., Samuelsson, B. and Haeggström, J.Z. (1996) Leukotriene A4 hydrolase, Mutation of tyrosine 378 allows conversion of Leukotriene A4 into an isomer of Leukotriene B4. J. Biol. Chem. 271, 24345-24348.

V. Mueller, M.J., Andberg, M., and Haeggström, J.Z. (1998) Analysis of the molecular mechanism of substrate-mediated inactivation of leukotriene A4 hydrolase. J. Biol. Chem. 273, 11570-11575.

Cover illustration: Structure of the catalytic zinc site including Tyr-378 and Tyr-383. Adopted from [183].

Grant support: This work was financially supported by the Swedish Medical Research Council, The European Union, Gustav V:s 80-års fond, the Foundation for Strategic Research, and the Deutsche Forsschungssgemeinschaft.

Abbreviations:

ATP Adenosine triphosphate COX Cyclooxygenase cPLA2 Cytosolic phospholipase A2 E. Coli Escherichia coli FLAP Five activating protein fMLP N-formyl-methionyl-leucyl-phenylalanine FPLC Fast protein liquid chromatography GC-MS Gas chromatography-mass spectrometry GSH Reduced HETE Hydroxyeicosatetraenoic acid HPETE Hydroperoxyeicosatetraenoic acid HPLC High performance liquid chromatography IL Interleukin Km Michaelis constant LTA4 Leukotriene A4, 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid LTB4 Leukotriene B4, 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid LTC4 Leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid LTD4 Leukotriene D4, 5(S)-hydroxy-6(R)-S-cysteinylglycyl-7,9-trans-11,14-cis-eicosatetraenoic acid LTE4 Leukotriene E4, 5(S)-hydroxy-6(R)-S-cysteinyl-7,9-trans-11,14-cis-eicosatetraenoic acid LX MMTS Methyl methanethiosulfonate PAF activating factor PCR Polymerase chain reaction PG RP-HPLC Reverse-phase high performance liquid chromatography SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis TX UV Ultraviolet 5-HETE 5(S)-Hydroxy-8,11,14-cis-6-trans-eicosatetraenoic acid 12-HETE 12(S)-Hydroxy-5,8,14-cis-10-trans-eicosatetraenoic acid 15-HETE 15(S)-Hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid

CONTENTS

1. INTRODUCTION 7 1.1. Metabolism of . 7 1.2. The . 7 1.3. Cytosolic phospholipase A2. 8 1.4. 5-Lipoxygenase. 8 1.4.1. Iron-binding ligands. 8 1.4.2. N-terminal β-barrel domain. 8 1.4.3. Promoter and gene structure. 9 1.4.4. 5-LO-deficient mice. 9 1.5. 5-Lipoxygenase activating protein (FLAP). 9 1.6. Leukotriene A4 hydrolase. 9 1.6.1. Substrate specificity and substrate mediated inactivation of LTA4 hydrolase. 10 1.6.2. Cloning and gene structure of LTA4 hydrolase. 10 1.6.3. LTA4 hydrolase contains a zinc site, which binds a single catalytic zinc. 10 1.6.4. Peptidase activity of LTA4 hydrolase. 10 1.6.5. LTA4 hydrolase belongs to the M1 family of metallopeptidases. 11 1.6.6. Mutagenesis of Glu-296 or Tyr-383 of LTA4 hydrolase selectively abrogates the peptidase activity. 11 1.6.7. Mutation of Tyr-383 provides evidence for an SN1 mechanism of the epoxide hydrolase reaction. 12 1.6.8. Proposed reaction mechanism for the peptidase activity. 12 1.6.9. Development of enzyme inhibitors. 12 1.6.10. Generation of LTA4 hydrolase deficient mice. 12 1.6.11. The crystal structure of LTA4 hydrolase. 13

1.7. . 13 1.8. Leukotriene biosynthesis occurs at the nuclear envelope. 14 1.9. Leukotriene receptors. 15 1.9.1. Receptors for LTB4, BLT1, and BLT2. 15 1.9.2. Receptors for cys-LTs, CysLT1, and CysLT2. 15

2. AIMS OF THE PRESENT INVESTIGATION 16

3. METHODOLOGY 16 3.1. Enzyme purification and expression of recombinant enzymes. 16 3.2. Site-directed mutagenesis. 16 3.3. Activity determinations. 16 3.3.1. Epoxide hydrolase activity. 16 3.3.2. Peptidase activity. 16

3.4. Chemical modification. 17 3.5. Preparation of suicide inactivated enzyme. 17 3.6. Peptide mapping. 17 3.7. Instrumental analysis. 17 3.7.1. Reverse-phase high performance liquid chromatography (RP-HPLC). 17 3.7.2. Gas chromatography / mass spectrometry (GC/MS). 17 3.7.3. Ultraviolet (UV) spectrophotometry. 17 3.7.4. Circular dichroism spectrometry (CD). 17

4. RESULTS 17

4.1. LTA4 hydrolase contains essential tyrosyl and arginyl residues at the active site as determined by chemical modification. 17

4.2. Identification of a central peptide segment in LTA4 hydrolase to which LTA4 binds during suicide inactivation. 17

5

4.3. LTA4 hydrolase can be protected from suicide inactivation by mutation of Tyr-378. 18

4.4. Mutants of Tyr-378 can hydrolyze LTA4 into a double-bond isomer of LTB4. 18

4.5. Evidence that the substrate-mediated inactivation of LTA4 hydrolase follows an affinity-labeling mechanism. 18

5. DISCUSSION 19

5.1. Identification of functional Tyr and Arg residues at the active site of LTA4 hydrolase. 19

5.2. Identification of molecular determinants of suicide inactivation of LTA4 hydrolase. 19 5.2.1. Identification of a peptide region involved in suicide inactivation. 20 5.2.2. Tyrosine 378 is a structural determinant for suicide inactivation. 21 5.2.3. Formation of a double-bond isomer of LTB4 by mutants of tyrosine 378. 21 5.2.4. Possible role of Tyr-378 in the hydrolysis of LTA4 into LTB4. 21

5.3. Molecular mechanism of substrate-mediated inactivation of LTA4 hydrolase. 22 5.3.1. The catalytically inactive mutant [E318A]LTA4 hydrolase is covalently modified by LTA4. 22 5.3.2. Covalent modification of a non-essential residue at the active site of LTA4 hydrolase. 22 5.3.3. LTA4 acts as an affinity label during self-catalyzed inactivation of LTA4 hydrolase. 22

5.4. Suicide inactivation of other enzymes in the cascade. 23 5.4.1. Cyclooxygenases. 23 5.4.2. PGI synthase and TX synthase. 23 5.4.3. 5-Lipoxygenase. 23 5.4.4. 12- and 15-lipoxygenase. 24

5.5. Comparison of structure-function data generated with biochemical techniques or X-ray crystallography. 24 5.5.1. The zinc site and catalytic residues. 24 5.5.2. A binding site for LTA4 and its implications for the epoxide hydrolase mechanism. 25 5.5.3. Location of Tyr-378 and Tyr-383. 25 5.5.4. Concluding remarks. 26

6. ACKNOWLEDGMENTS 27

7. REFERENCES 27

8. ORIGINAL PUBLICATIONS

6

Introduction

1. INTRODUCTION

Inflammation is an important pathophysiological charac- 1.2. The leukotrienes. teristic of a large number of severe endemic diseases with Leukotrienes (LT), a group of lipid compounds with po- the potential to afflict almost all tissues and organ sys- tent biological activities related to inflammatory and al- tems. Some of these illnesses may be fatal whereas others lergic disorders, were discovered more than twenty years are chronically progressive and crippling. In the microen- ago [14,126]. These compounds are derived from the vironment, the development and maintenance of inflam- metabolism of polyunsaturated fatty acids, particularly mation are governed by a complex network of cellular and arachidonic acid [165] (Fig. 1). In two consecutive reac- humoral factors. Among these are the , a class tions, catalyzed by 5-LO, arachidonic acid is transformed of structurally related paracrine hormones derived from into LTA4, the key intermediate in leukotriene biosynthe- the oxidative metabolism of arachidonic acid that includes sis. This highly unstable epoxide may either undergo the (PG), the (TX), the enzymatic hydrolysis into the dihydroxy acid LTB4 or be leukotrienes (LT), and the (LX). conjugated with glutathione, to form LTC4. The latter compound, together with its metabolites LTD4 and LTE4, 1.1. Metabolism of arachidonic acid. are referred to as the cysteinyl-containing leukotrienes The PGs and TXs are synthesized via the action of two (cys-LTs). cyclooxygenase (COX) enzymes, the stably expressed As indicated by the name leukotriene, these com- COX-1 and the more recently discovered and inducible pounds were originally isolated from leukocytes [14,126]. variant COX-2 [178]. These two enzymes are believed to Bone marrow derived cells are the main producers, par- play isozyme-specific roles in house-keeping functions ticularly polymorphonuclear leukocytes, monocytes as and host defense/inflammation, respectively. Both COX-1 well as tissue-bound and mast cells. Certain and COX-2 generate the common intermediate PGH2 cells, e.g. , , and mast cells mainly from which all other PGs and TXs are biosynthesized via synthesize cys-LTs, whereas others, e.g. and down-stream synthases, in particular the highly specific macrophages predominantly generate LTB4. PGI synthase and TX-synthase, as well as PGD syn- Leukotrienes possess a wide range of biological ac- thase(s), PGE synthase(s), and PGF synthase(s). These tivities elicited via specific cell surface receptors [99,165]. PGs and TXs act via a family of G-protein coupled recep- LTB4 is a very potent chemotaxin for neutrophils and tors [128]. recruits inflammatory cells to the site of injury. This com- In contrast, both the LTs and LXs are formed along 5- pound also induces chemokinesis and increases leukocyte lipoxygenase (5-LO) dependent pathways, involving the adhesion to the endothelial cells of the vessel wall. The common intermediate LTA4 (see section 1.2.). In mam- cys-LTs on the other hand are potent constrictors of mals, 12- and 15- (12-LO, 15-LO) also smooth muscle, particularly in the airways where they metabolize arachidonic acid, primarily into hydroperoxy- elicit . In the microcirculation, they (HPETEs) and hydroxy-eicosatetraenoic acids (HETEs), evoke constriction of arterioles and increase the perme- the biological significance of which are not fully under- ability of the postcapillary venules leading to extra- stood [43]. vasation of plasma. Due to their potent biological activi-

Fig. 1. Biosynthesis of leukotrienes.

7 Martin J. Mueller ties, leukotrienes are strong candidates as chemical me- riene biosynthesis, the role of cPLA2 in this process was diators in a number of inflammatory and allergic disor- for long questioned. However, knock-out mice lacking the ders, e.g. rheumatoid arthritis, inflammatory bowel dis- cPLA2 gene have been generated and the peritoneal ease, and bronchial [99]. In fact, several drugs macrophages of the cPLA2 deficient mice generated nei- have been developed which block the synthesis and action ther prostaglandins nor leukotrienes in response to ade- of leukotrienes [35]. Some of these agents, in particular quate stimuli [12,186]. Hence, cPLA2 is certainly a criti- ® the CysLT1- antagonists (Singulair ) cal lipase involved in leukotriene biosynthesis. ® and (Accolate ) are presently available as The promoter of the human cPLA2 gene has been novel anti-asthmatic drugs. cloned and partially characterized [121]. It has typical features of a house-keeping gene with no TATA or CAAT 1.3. Cytosolic phospholipase A2. box, although atypical in that it is not GC rich and lacks In the living cell the majority of arachidonic acid is esteri- Sp1/Egr-1 sites. Instead, it has a long stretch of CA re- fied in the sn-2 position of phospholipids. In order to peats and a polypyrimidine tract. The 5'-flanking region increase the levels of free arachidonic acid available for also contains a putative composite AP-1 site and gluko- leukotriene biosynthesis, a phospholipase is required to corticoid response element (GRE). release the fatty acid from the cell membranes (Fig. 1). Phospholipases comprise a large family of enzymes [29], 1.4. 5-Lipoxygenase. several of which have been implicated in leukotriene bio- 5-Lipoxygenase (5-LO) (EC 1.13.11.34) is a soluble synthesis. Although it has been disputed [109], it is now monomeric enzyme with a molecular mass of about 78 clear that the group IV, high molecular weight, cytosolic kDa [161], which contains one atom of non-heme iron, phosholipase A2 (cPLA2) (EC 3.1.1.4.) plays a key role in believed to be of importance for catalysis [149]. The en- the generation of arachidonic acid for leukotriene biosyn- zyme catalyzes the first two steps in leukotriene biosyn- thesis. This enzyme has a molecular mass of 85 kDa and thesis [160,176]. In the first reaction, free arachidonic is activated and translocates to membranes in response to acid is oxygenated into the hydroperoxide 5-HPETE, submicromolar levels of Ca2+ [48,171]. Moreover, this which is subsequently dehydrated to yield the unstable enzyme selectively hydrolyzes arachidonic acid esterified epoxide intermediate LTA4. For maximal activity, 5-LO 2+ in the sn-2 position of phospholipids. Cytosolic PLA2 was requires both Ca and ATP and is stimulated by lipid purified to homogeneity from U937 cells and molecular hydroperoxides and phosphatidylcholine [153,161,163]. cloning revealed a cDNA which predicts a protein of 748 amino acids (initial Met excluded) [24,25,85]. A protein 1.4.1. Iron-binding ligands - The cDNA encoding hu- domain, located in the N-terminal part of cPLA2, was man 5-LO has been cloned and sequenced and the de- shown to associate with membrane vesicles in response to duced amino acid sequence revealed the presence of six Ca2+. This domain contains a segment with homology to histidines, canonical in plant and animal lipoxygenases the constant region 2 (C2 domain) of protein kinase C [34,111]. Mutagenetic replacements of these histidines in (PKC), believed to be of importance for translocation of human 5-LO, in combination with iron determinations of PKC. Thus, cPLA2, in its amino terminal part, contains a the purified recombinant proteins, demonstrated that His- putative Ca2+-dependent phospholipid binding (CaLB or 372 and His-550 are iron ligands, whereas the iron- C2) domain. Experiments with recombinant DNA tech- binding and/or catalytic function of His-367 remains un- nology verified the regulatory role of this protein segment certain [72,129,151,208,209]. From the three-dimensional for the Ca2+-dependent binding of phospholipids, and also structure of soybean lipoxygenase-1, which corroborated showed that the active site is localized in the C-terminal and extended the information obtained by mutagenesis portion of the polypeptide chain [127]. Recently, the solu- [15,120], it was discovered that the C-terminal isoleucin, tion structure of the C2 domain of cPLA2 demonstrated Ile-673 in human 5-LO, also functions as an iron that it is a β-sandwich structure that binds two Ca2+ ions [61]. Two other amino acids in 5-LO, viz. Glu-376 and and preferentially interacts with phosphocholine head- Gln-558 are functionally important, since mutagenetic 2+ groups [201]. The Ca -dependent translocation of cPLA2 replacements of these residues result in complete loss of to ER and nuclear membranes with concomitant enzyme enzyme activity [72,209]. activation [48,171] requires a functional C2 domain [46]. In addition, the complete crystal structure of cPLA2 at 2.5 1.4.2. N-terminal β-barrel domain - The first crystal Å resolution has been determined and revealed an unusual structure of a mammalian lipoxygenase, viz. rabbit 15-LO, catalytic Ser-Asp dyad, located in a deep cleft at the cen- was determined in 1997 [47]. Interestingly, the architec- ter of a hydrophobic funnel, which cleaves arachidonoyl ture of the substrate-binding site clarified the molecular phospholipids [30]. Interestingly, the structure also re- mechanism for the positional specificity for oxygenation vealed a flexible lid that must move to allow substrate of arachidonic acid displayed by 5-, 12- and 15-LO. Thus, access to the active site. Cytosolic PLA2 is also regulated the depth and width of the substrate binding pocket ap- post-translationally by phosphorylation, particularly at pears to be critical such that a shallow pocket puts the Ser-505, which leads to activation in the presence of Ca2+ catalytic iron close to C-15, whereas a deep pocket puts it [28,100]. close to C-5. Furthermore, rabbit 15-LO contains an N- Since most of the ligands that have been used to acti- terminal β-barrel domain, a structure also found in the C- vate cPLA2 are related to PG synthesis, and not leukot- terminal domain of lipases. The role of this domain for

8

Introduction lipoxygenases is presently unclear but for 5-LO it has augments phagocytosis mediated by either the been shown to bind Ca2+ and presumably facilitates the Fc or complement receptor [108]. Moreover, 5-LO defi- association of 5-LO with membranes during catalysis [60]. cient mice exhibit a reduced airway reactivity in response This β-barrel domain has also been shown to be essential to methacholine and lower levels of serum immuno- for translocation to the nuclear membrane, a typical fea- globulins [71]. ture of 5-LO [22]. Moreover, using in-gel kinase assays, 5-LO was recently shown to be a substrate for p38 kinase- 1.5. 5-Lipoxygenase activating protein (FLAP). dependent MAPKAP kinases, suggesting that phosphory- In early work by Rouzer et al., carried out with intact lation may be one additional factor, which determines 5- cells, it was shown that 5-LO becomes activated and trans- LO translocation and enzyme activity [191] (see further locates to membranes in response to Ca2+, a process ac- section 1.8.). companied by catalysis and enzyme inactivation [159,162,197]. Subsequently, it was demonstrated that 1.4.3. Promoter and gene structure - The gene encod- cellular 5-LO activity is dependent on a small membrane ing human 5-LO is located on chromosome 10 and spans protein, termed five lipoxygenase activating protein more than 80 kb of DNA consisting of 14 exons divided (FLAP). This protein was discovered through the inhibi- by 13 introns [43,44]. The promoter region lacks a typical tory action of a drug, MK-886, on leukotriene biosynthe- TATA or CCAT box but contains a number of GC boxes, sis in intact cells [158]. MK-886 could bind to this mem- potential binding sites for the transcription factors Sp1 brane protein and thereby prevent and revert translocation and Egr-1. Thus, the promoter structure resembles those of 5-LO, which in turn suggested direct interactions with of so called house-keeping genes, which are constitutively 5-LO. FLAP was purified by means of affinity chromatog- expressed in many cells and tissues. This was a surprising raphy, characterized, and cloned [33,117]. The amino acid finding since 5-LO activity has been detected almost ex- sequence showed that it is a unique protein with three clusively in bone marrow derived cells. putative transmembrane domains. Transfection experi- The promoter of the 5-LO gene has been found to con- ments of osteosarcoma cells with 5-LO cDNA alone or tain naturally occurring mutations with respect to the together with FLAP cDNA demonstrated that FLAP is number of GC boxes, which in turn appears to influence essential for leukotriene biosynthesis in intact cells [33]. the expression of the 5-LO gene, as judged from pro- Although the mechanism of action for FLAP is not fully moter-reporter activities in cell culture [70]. It was sug- understood, it has been suggested that the role of FLAP is gested that this family of polymorphisms could be related to present or transfer arachidonic acid to 5-LO. Thus, to the inter-individual differences in clinical effects of 5- FLAP was shown to be an arachidonate binding protein LO inhibitors that are observed among asthma patients. In and this binding could be competed by compounds such fact, a pharmacogenetic association between 5-LO pro- as MK-886 [105]. FLAP also stimulates the utilization of moter genotype and the response to anti-asthma treatment arachidonic acid by 5-LO and increases the efficiency was recently demonstrated [36]. with which 5-LO converts 5-HPETE into LTA4 [1]. How- ever, in spite of many evidences suggesting a close rela- 1.4.4. 5-LO-deficient mice - The biological role of 5- tion between 5-LO and FLAP, a direct physical interac- LO and its products has been studied by gene targeting tion has never been demonstrated, which in turn indicates [23]. 5-LO-deficient mice are more resistant to lethal that other functional links exist between the two proteins. effects of shock induced by PAF and also show a marked The FLAP gene has been cloned and spans more than reduction in the ear inflammatory response to exogenous 31 kb on chromosome 13 [42,82]. It is divided into five arachidonic acid. Interestingly, the inflammatory response small exons separated by four large introns and the pro- induced by arachidonic acid could be virtually eliminated moter region contains a possible TATA box as well as a by the COX inhibitor indomethacin in 5-LO-deficient potential GRE and AP-2 binding site. mice, but not in normal animals, suggesting links between The role of leukotrienes as inflammatory mediators PGs and 5-LO products during inflammatory reactions has also been corroborated in studies of FLAP deficient [51]. Apparently, there are significant differences between mice. Like the 5-LO (-/-) mice, these animals show a mouse strains in their relative susceptibility to PG- or LT- blunted inflammatory response to topical arachidonic dependent inflammatory responses [49]. Interestingly, acid, have increased resistance to PAF induced shock, and when a strain of MRL-lpr/lpr mice, which spontaneously respond with less edema in zymosan-induced peritonitis develop symptoms of autoimmune disease with increased [20]. Furthermore, the severity of collagen induced arthri- mortality among female mice, were crossed with 5-LO (-/- tis is substantially reduced in FLAP (-/-) mice, indicating ) mice, the sex-related survival differences were abol- a role for leukotrienes in this model of inflammation [53]. ished. These findings suggest that the presence of a func- tional 5-LO gene confers a survival advantage on male 1.6. Leukotriene A4 hydrolase. MRL-lpr/lpr mice and that, when 5-LO function is LTA4 hydrolase (EC 3.3.2.6) is a soluble and monomeric blocked, this advantage is lost [50]. It has also been enzyme, which catalyzes the hydrolysis of the unstable shown that 5-LO null mice are more susceptible to infec- epoxide intermediate LTA4 into LTB4 (Fig. 1). The pro- tions with Klebsiella pnemoniae, in line with a role for 5- tein is widely distributed and has been detected in almost LO and its products in anti-microbial host defense all mammalian cells, organs, and tissues examined [192]. [5,172]. In fact, it was recently demonstrated that LTB4 Among cellular elements of the blood, neutrophils, mono-

9 Martin J. Mueller cytes, lymphocytes, and erythrocytes are rich sources of The gene structure of human LTA4 hydrolase has also the enzyme, whereas eosinophils have low levels and been determined [106]. It is a single copy gene with a size basophils and seem to lack LTA4 hydrolase. The of > 35 kbp. The coding sequence is divided into 19 exons enzyme is found even in cells lacking 5-LO activity and ranging in size from 24 to 312 bp. The human LTA4 hy- thus the ability to provide the substrate LTA4. Examples drolase gene was mapped to chromosome 12q22, using of such cells are erythrocytes, T-cell lines, fibroblasts, fluorescence in situ hybridization. The putative promoter endothelial cells, keratinocytes, and airway epithelial cells region (approx. 4 kbp) contains a phorbol-ester response [192]. The subcellular localization of LTA4 hydrolase has element (AP-2) and two xenobiotic-response elements never been thoroughly investigated but it is assumed that (XRE) but no definitive TATA box. The significance of the activity resides in the cytosol, although a membrane these putative cis-elements has not been determined. bound activity has also been reported [55]. LTA4 hydrolase has been purified from a variety of 1.6.3. LTA4 hydrolase contains a zinc site which binds mammalian sources and, in general, the catalytic proper- a single catalytic zinc - When the amino acid sequence of ties and physicochemical characteristics are similar for the LTA4 hydrolase was compared to that of rat kidney amin- different enzymes [192]. LTA4 hydrolase has also been opeptidase M it was found that the two proteins were isolated from the African claw toad Xenopus laevis [180]. weakly homologous to each other and to several other This enzyme, in contrast to the mammalian counterparts, zinc hydrolases [104]. The similarity was higher over a 6 has the capacity to generate not only LTB4 but also ∆ - short segment of the respective proteins, which contains a 8 trans-∆ -cis LTB4 from the substrate LTA4 [179]. consensus sequence for a catalytic zinc site (H-E-(X)1-3- Very little is known about the cellular regulation of H-(X)18-120-E) [188]. This observation suggested that LTA4 hydrolase. Certain cytokines, in particular IL-4 and LTA4 hydrolase is a zinc-containing enzyme and that His- IL-13, have been reported to modulate the cellular expres- 295, His-299, and Glu-318 were likely candidates to be sion of the enzyme. However, these two cytokines led to the zinc binding ligands. Samples of purified enzyme were opposite effects in monocytes and polymorphonuclear analyzed by atomic absorption spectrometry, which leukocytes, respectively, which makes it difficult to draw showed that LTA4 hydrolase contains one mole of any certain conclusions [122,207]. Furthermore, it has zinc/mole of protein [57,119]. In addition, enzyme activ- been reported that LTA4 hydrolase isolated from endothe- ity could be inactivated by the zinc chelator 1,10- lial cells is inhibited by phosphorylation, specifically phenanthroline. This inactivated protein preparation did affecting the enzyme’s ability to convert LTA4 into LTB4 not contain any significant amounts of zinc and thus rep- [164]. resented the apoenzyme of LTA4 hydrolase. The enzyme activity could be restored by addition of stoichiometric 1.6.1. Substrate specificity and substrate mediated in- amounts of zinc to the apoenzyme and hence, the primary activation of LTA4 hydrolase - LTA4 hydrolase has a function of the metal seems to be catalytic rather than narrow substrate specificity [136] and requires a 5,6- structural, in agreement with the organization of the zinc trans-epoxide with a free carboxylic acid at C-1 of the binding ligands. fatty acid [40,114]. The double-bond geometry of the The three proposed zinc binding ligands in LTA4 substrate is also essential for catalysis and LTA3, which hydrolase have been verified by site-directed mutagenesis 14 lacks the ∆ double bond, is a poor substrate for LTA4 [116]. Thus, His-295, His-299, and Glu-318 were indi- hydrolase [38]. vidually replaced by Tyr, Tyr, and Gln, respectively, none The enzyme is inactivated and covalently modified by of which can bind zinc. The three mutants were all found its substrate LTA4 and, even more effectively, by the to be enzymatically inactive and when they were subjected structural isomers LTA3, LTA5, and the methyl ester of to atomic absorption spectrometry, only minute amounts LTA4 [38,136]. Kinetic analysis has shown that the inac- of zinc (< 0.07 eq.) could be detected. tivation is directly coupled to catalysis and proportional to product formation [138]. Electrospray mass spectrometry 1.6.4. Peptidase activity of LTA4 hydrolase - The iden- showed a shift in molecular weight of inactivated enzyme tification of LTA4 hydrolase as a member of a family of compatible with the coupling of LTA4 in a 1:1 zinc metalloproteases suggested that the enzyme could stoichiometry between lipid and protein [138]. The com- possess a peptide cleaving activity, in addition to its epox- petitive inhibitor bestatin prevents the covalent binding of ide hydrolase activity (the conversion of LTA4 into LTA4 to the enzyme, indicating that it occurs at the active LTB4). Indeed it was found that LTA4 hydrolase could site [37,138]. For more details on the mechanism of this hydrolyze a number of synthetic chromogenic substrates inactivation process, see Papers II, III, and V. [58,119]. Also the peptidase activity could be inhibited by the chelator 1,10-phenanthroline and the activity of the 1.6.2. Cloning and gene structure of LTA4 hydrolase - apoenzyme was regained by stoichiometric amounts of The cDNAs encoding the human, mouse, rat, and guinea- zinc. Apparently, the presence of a zinc-binding motif in a pig enzymes have been cloned, sequenced, and expressed protein is only indicative of a peptide cleaving activity. in E. coli [56]. They all contain 610 amino acid residues Thus, preparations of aminopeptidases from porcine and (initial Met excluded), with a calculated molecular mass hog kidney as well as bovine intestine, do not convert of 69 kDa. The amino acid sequences are highly con- LTA4 into LTB4 [58,119]. served and exhibit > 90% identity between these species.

10

Introduction

Among a panel of different amino acids coupled to either 4-nitroaniline or ß-naphtylamine, the alanine, argin- ine and proline derivatives were the most effectively hy- drolyzed substrates [119,193]. When the peptide substrate specificity was systematically investigated, the enzyme was found to hydrolyze the N-terminal arginine from the tripeptides Arg-Gly-Asp, Arg-Gly-Gly, and Arg-His-Phe with specificity constants (kcat/Km) in the same range as the lipid substrate LTA4, suggesting that LTA4 hydrolase is an arginyl tripeptidase [139]. Although several opioid peptides have been shown to be hydrolyzed by LTA4 hydrolase no strong candidate of a natural substrate has yet been presented [52,133]. The peptidase activity of LTA4 hydrolase is stimulated by several monovalent anions [193]. Thiocyanate is the most effective followed by chloride and bromide ions. The stimulation is mediated by an increase in turnover without significantly affecting the Km for the substrate. The stimulatory effect of chloride on the reaction veloci- Fig. 2. Structural and functional features of LTA4 ties appears to obey saturation kinetics indicating the hydrolase. presence of an anion binding site, with an apparent affin- ity constant (KA) for chloride ions of 100 mM. Notably, no anion-stimulation is observed for the epoxide hy- As an exception which confirms the rule, an LTA4 hy- drolase activity. drolase was recently cloned from Saccharomyces cere- Albumin has been reported to be another selective visiae and characterized as a bifunctional enzyme with an stimulus for the peptidase activity, which is increased 3- anion stimulated leucyl aminopeptidase activity. Interest- to 24-fold, depending on the type of substrate. In this case, ingly, this enzyme exhibits an epoxide hydrolase activity the stimulatory effect is due to a decreased Km [137]. The in which LTA4 is used as substrate to produce 5S,6S- 6 8 selective stimulation of the peptidase activity of LTA4 DHETE, LTB4, and ∆ -trans-∆ -cis-LTB4 [86,87]. It was hydrolase by chloride and albumin could represent a mode also demonstrated that the S. cerevisiae LTA4 hydrolase of enzyme regulation. Considering the differences in chlo- possesses a lipid-binding pocket, which mediates stimula- ride concentration between the intracellular and extracel- tion of the peptidase activity when occupied by LTA4. lular compartments and the fact that albumin is the pre- Thus, it appears highly likely that the yeast enzyme is an dominant plasma protein, one can speculate that the pepti- evolutionary distant ancestor of the mammalian LTA4 dase activity of LTA4 hydrolase may only proceed outside hydrolases. the cell, whereas the epoxide hydrolase activity may oper- ate on either side of the cell membrane. Interestingly, 1.6.6. Mutagenesis of Glu-296 or Tyr-383 of LTA4 hy- LTA4 hydrolase activity has been detected in plasma of drolase selectively abrogates the peptidase activity - The several mammals and high levels of the enzyme have also zinc site of LTA4 hydrolase contains a conserved Glu been found in human bronchoalveolar lavage fluid residue located next to the first zinc binding ligand, a [40,123]. feature found in several zinc proteases or mono zinc ami- nopeptidases. From X-ray crystallographic studies on 1.6.5. LTA4 hydrolase belongs to the M1 family of thermolysin, the corresponding residue (Glu-143) has metallopeptidases - Based on its zinc signature, sequence been suggested to serve as a general base in the catalytic homology, and aminopeptidase activity, LTA4 hydrolase mechanism [83,143]. When the counterpart in LTA4 hy- has been classified as a member of the M1 family of met- drolase, Glu-296, was substituted for a Gln, Asp, Asn, or allopeptidases [6]. Thus, LTA4 hydrolase is distantly Ala residue by site-directed mutagenesis, the mutated related to many other zinc proteases and aminopeptidases proteins [E296Q], [E296D], [E296N], and [E296A]-LTA4 that are present in a variety of organisms from bacteria to hydrolase exhibited epoxide hydrolase activities between mammals, including human enzymes such as aminopepti- 150 and 15% of wild type enzyme, respectively dase A (APA), aminopeptidase B (APB), and aminopepti- [4,118,196]. In contrast, all mutants were essentially de- dase N (APN). For most of these proteins, the level of void of peptidase activity (< 0.25% of wild type enzyme). identity (similarity) with LTA4 hydrolase is low and es- Thus, Glu-296 seems to be essential for the peptidase sentially confined to the zinc binding site, whereas in activity, whereas the hydrolysis of LTA4 into LTB4 may some cases, e.g., an arginyl aminopeptidase in C. elegans proceed also with other amino acid residues in this posi- [7], the degree of homology is higher and more evenly tion. distributed along the primary structures. However, these Sequence comparisons of LTA4 hydrolase and amin- proteins are solely proteases or aminopeptidases without opeptidase M revealed the presence of a conserved an epoxide hydrolase activity. nonapeptide, suggested to be a proton donor motif. In LTA4 hydrolase, Tyr-383 was proposed to serve as a

11 Martin J. Mueller proton donor [118]. This residue was replaced for either a similar mechanism, Glu-296 would serve as the base and Phe, His or Glu by site-directed mutagenesis [11]. All Tyr-383 as the proton donor. three mutants [Y383F], [Y383H], and [Y383Q]LTA4 hydrolase lacked significant peptidase activities, in line 1.6.9. Development of enzyme inhibitors - The discov- with a role for Tyr-383 as a proton donor. The epoxide ery that LTA4 hydrolase belongs to a family of zinc prote- hydrolase activity was reduced, but still clearly measur- ases opened up new possibilities in the search for enzyme able and a kinetic analysis of [Y383Q], revealed a Km inhibitors. Bestatin and are inhibitors of amin- value for LTA4 which was ten-fold higher than for wild opeptidases and angiotensin converting enzyme, respec- type enzyme which in part could explain the reduced tively, and were found to be effective inhibitors also of specific activity at lower concentrations of LTA4. More- LTA4 hydrolase [141]. Furthermore, kelatorphan, a over, it suggests that Tyr-383 could be involved in sub- known inhibitor of enkephalin degrading enzymes is a strate (LTA4) binding. potent inhibitor of LTA4 hydrolase [144] and a class of ω- The results obtained from mutagenesis of Glu-296 and [(ω-arylalkyl)aryl]alkanoic acids were reported to inhibit Tyr-383 suggest that the two enzymatic activities are LTA4 hydrolase in the low µM range, one of which was exerted via overlapping but not identical active sites as metabolically stable after oral administration to rats [90]. depicted in Fig. 2. The role of Tyr-378 is described in Several laboratories have developed more powerful Papers III, IV, and V. and selective compounds, based on proposed reaction mechanisms and inhibitor-enzyme interactions for other 1.6.7. Mutation of Tyr-383 provides evidence for an zinc hydrolases. For example, an α-keto-β-amino ester, a SN1 mechanism of the epoxide hydrolase reaction - Fur- thioamine, and an amino hydroxamic acid were synthe- ther investigation of the catalytic properties of mutants in sized and found to be effective tight-binding inhibitors position 383, revealed the formation of large quantities of with IC50 values in the low µM to nM range [68,194,206]. a novel metabolite of LTA4 structurally identified as These three compounds were also potent and selective 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid inhibitors of LTB4 biosynthesis in intact human leuko- (5S,6S-DHETE), in addition to the expected LTB4 [10]. cytes [68,194]. Interestingly, the stereochemistry of 5S,6S-DHETE im- A series of inhibitors of LTA4 hydrolase have been plies an SN1 mechanism in its formation involving a car- developed by Searle, e.g., SC-57461, N-methyl-N-[3-[4- bocation intermediate, which in turn indicates that conver- (phenylmethyl)-phenoxy]propyl]-β-alanine which blocks sion of LTA4 into LTB4 proceeds according to the same ionophore-induced LTB4 production in human whole mechanism (see further section 5.5.2.). blood with an IC50 of 49 nM [205] and more recently derivatives of SC-22716, in particular 2-[4-[4-[2-(1- 1.6.8. Proposed reaction mechanism for the peptidase pyrrolidinyl)ethoxy]phenoxy]phenyl]-oxazole [145]. Both activity - In a catalytic model suggested for thermolysin, compounds are orally active and the former structure has the reaction is initiated by a water molecule which is dis- also shown promising results in an animal model of colitis placed from the zinc atom by the carbonyl oxygen of the [177]. substrate and then polarized by the carboxylate of a glu- tamic acid to promote an attack on the carbonyl carbon of 1.6.10. Generation of LTA4 hydrolase deficient mice - the scissile peptide bond (Fig. 3). Simultaneously, a pro- Mice deficient in LTA4 hydrolase, and thus the ability to ton is transferred to the nitrogen of the peptide bond from convert LTA4 into LTB4, have been generated by targeted an adjacent amino acid [83,143]. Assuming that the pepti- gene disruption [21]. These mice develop normally and dase activity of LTA4 hydrolase proceeds according to a

Fig. 3. Proposed reaction mechanism for the peptidase activity of LTA4 hydrolase.

12

Introduction are healthy. Analysis of their reactivity against various The complete gene for human LTC4 synthase has also proinflammatory stimuli, revealed that LTA4 hydrolase is been cloned and sequenced [147]. This gene is located on required for the formation of LTB4 during an in vivo in- chromosome 5, spans about 2.5 kb and has a structure flammatory reaction. Comparing the phenotype of these similar to the FLAP gene. Thus, it contains five exons and mice with that of 5-LO (-/-) mice, allowed a delineation of has exon-intron junctions that align identically with those the relative contribution of LTB4 and cys-LTs, respec- of FLAP. The promoter region of the LTC4 synthase gene tively, to a specific inflammatory response. Thus, LTB4 is contains several potential cis-elements including, Sp1, responsible for the characteristic influx of neutrophils, AP-1 and AP-2. Further promoter characterization re- which follows topical application of arachidonic acid and vealed that an Sp1 site and a putative initiator element contributes to the vascular changes observed in this in- (Inr) are involved in non-cell-specific expression, whereas flammatory model. In zymosan-A induced peritonitis, a Kruppel-like transcription factor and Sp1 are implied in LTB4 modulates only the cellular component of the re- cell-specific regulation of the LTC4 synthase gene [210]. sponse whereas LTC4 appears to be responsible for the In the monocyte-like cell line THP-1, however, both Sp1 plasma protein extravasation. Moreover, LTA4 hydrolase and Sp3 were shown to regulate LTC4 synthase gene was recently shown to be upregulated in the hearts of expression, and the former transcription factor apparently angiotensin II-induced hypertensive rats, thus providing acts via a different Sp1 site [174]. further evidence for a role of LTA4 hydrolase in inflam- Several in vitro studies have addressed the issue of matory reactions in vivo [73]. Of note, LTA4 hydrolase cytokine regulation of the LTC4 synthase gene, e.g., in (-/-) mice are resistant to the lethal effects of systemic mast cells and eosinophilic cell lines [124,125,173]. As is shock induced by PAF, thus identifying LTB4 as a key often the case with cytokine effects, induction of the LTC4 mediator of this reaction. synthase gene depends on the priming effects of other cytokines as well as the combinations and sequences of 1.6.11. The crystal structure of LTA4 hydrolase - The their addition. Thus, after priming of mouse bone marrow- human LTA4 hydrolase has been crystallized in the pres- derived mast cells with c-kit ligand and IL-10, IL-3 ence of the competitive inhibitor bestatin [183]. The three upregulates the 5-LO pathway enzymes including LTC4 dimensional structure revealed a protein with three do- synthase [124], whereas IL-4 has a counter-regulatory mains which together form a deep cleft harboring the zinc effect in this system [125]. In contrast, IL-4 triggers a site. The catalytic domain is structurally very similar to profound induction of LTC4 synthase in human mast cells, thermolysin. A more detailed description of the enzyme derived from cord blood progenitors with SCF and IL-6, structure is presented in section 5.5. and in this system IL-3 and IL-5 has a priming effect [69]. It has also been shown that TGFβ increases LTC4 syn- 1.7. Leukotriene C4 synthase. thase expression in THP-1 cells [156]. Leukotriene C4 synthase (EC 2.5.1.37) catalyzes the Interestingly, overexpression of LTC4 synthase was committed step in the biosynthesis of cys-LTs through observed in bronchial biopsies from patients of aspirin- conjugation of LTA4 with glutathione (Fig. 1). The en- intolerant asthma [26], suggesting a pathophysiological zyme is a membrane bound homodimer with a subunit role for LTC4 synthase in this disease. Analysis of the molecular mass of 18 kDa [130,131,146]. It is stimulated LTC4 synthase gene promoter by single-strand by divalent cations, particularly Mg2+, as well as phos- conformation polymorphism (SSCP) revealed the phatidylcholine, whereas reduced glutathione is required presence of a A-444C transversion. This mutation was for stability. LTC4 synthase has been cloned and se- found in higher frequency in patients with aspirin- quenced [92,190]. Two consensus sequences for protein intolerant asthma as compared to patients with aspirin– kinase C phosphorylation were found and subsequent tolerant asthma or healthy controls [167]. The A-444C studies have shown that phosphorylation, which appears transversion creates an additional core motif for the to occur in a cell-specific fashion, reduces the LTC4 syn- transcription factor AP-2, suggesting that it may lead to an thase activity [3,54]. Surprisingly, sequence comparisons increased expression of LTC4 synthase [166,167]. of LTC4 synthase and FLAP demonstrated a 31% identity However, in a recent study, carried out within the between the two proteins. In addition, recent work has American population, no significant correlation was found identified two microsomal glutathione transferases between the LTC4 synthase promoter polymorphism and (MGST2 and MGST3), which both possess LTC4 syn- aspirin-intolerant asthma [189]. thase activity and exhibit a high degree of similarity to Only very recently, LTC4 synthase deficient animals both LTC4 synthase and FLAP [74,75]. Sequence com- were generated by targeted gene-disruption [79]. The parisons between various members of the LTC4 syn- LTC4 synthase (-/-) mice developed normally and were thase/FLAP/MGST gene family have identified conserved fertile. Bone marrow-derived mast cells from the LTC4 residues of potential functional importance and subse- synthase null mice exhibited a similar capacity for exocy- quent mutational analysis suggested that Arg-51 and Tyr- tosis and generation of PGD2, LTB4, and 5-HETE, as did 93 in human LTC4 synthase are both essential for cataly- cells from normal animals. In agreement with the results sis. The guanidinium moiety of Arg-51 is believed to open obtained with LTA4 hydrolase (-/-) mice, the LTC4 syn- the epoxide whereas Tyr-93 acts as a base to generate a thase (-/-) mice displayed a reduced plasma protein extra- glutathione thiolate anion for attack at C-6 of LTA4 [93].

13 Martin J. Mueller

1. TRANSLOCATION 2. BIOSYNTHESIS

Cell membrane Cell membrane

2+ Ca2+ Ca 2+ Ca2+ Ca Ca2+ Ca2+ cPLA2 cPLA 2+ 2 Ca AA

Nucleus Nucleus FLAP FLAP AA

2+ 5-LO Ca 5-LO 2+ Ca2+ Ca Ca2+ LTC4 LTC4 Ca2+ synthase synthase Ca2+ LTA Ca2+ 4

LTA4 LTA4 hydrolase hydrolase

LTB4 LTC4

Fig. 4. Activation and translocation of 5-LO and cPLA2 to the nuclear membrane followed by leukotriene biosynthesis.

vasation in zymosan A-induced peritoneal inflammation. inhibitor , it was demonstrated that translocation In addition, these mice were less prone to develop passive and catalysis are not necessarily coupled and that translo- cutaneous anaphylaxis. Certainly, the LTC4 synthase cation/membrane association can alter the substrate speci- deficient mice will be a useful tool for further studies of ficity of 5-LO and increase the efficiency of LTA4 biosyn- the biological role of LTC4 in physiological as well as thesis [67]. Of particular interest was the discovery that pathophysiological conditions. For instance, further in- FLAP is localized to the nuclear envelope of resting and sights may be gained regarding the recently described role activated neutrophils and that 5-LO, upon cell activation, of LTC4 in the primary immune response involving mobi- translocates to the same compartment [199]. Translocation lization of dendritic cells to lymph nodes [157], or the of 5-LO also occurs in cells that do not express FLAP, LTC4 deficiency associated with pediatric neurodegenera- and has therefore been suggested to be a FLAP- tive diseases [112,113]. independent process [80]. Furthermore, 5-LO has been shown to associate with growth factor receptor-binding 1.8. Leukotriene biosynthesis occurs at the nuclear protein 2 (Grb2), an ”adaptor” protein for tyrosine kinase- envelope. mediated , through Src homology 3 (SH3) In addition to what has been discussed above, all the en- domain interactions [95]. SH3 interactions regulate the zyme components of the leukotriene cascade are regulated assembly of protein complexes involved in cell signaling at multiple levels by a complex network of signaling and cytoskeletal organization, and may form the molecular mechanisms, e.g., cytokines, lipid mediators, and trans- basis for 5-LO translocation and compartmentalization. In cellular metabolism [9]. It is not within the scope of this agreement with this hypothesis, inhibitors of tyrosine thesis to cover these intricate relationships and instead, the kinase activity, a determinant of SH3 interactions, also intracellular compartmentalization of 5-LO and the as- inhibited the catalytic activity of 5-LO and its transloca- sembly of a biosynthetic complex at the nuclear mem- tion during cellular activation [98]. In addition, an internal brane, is discussed in some detail (see Fig. 4). bipartite nuclear localization sequence, spanning Arg-638 Early studies showed that upon cell stimulation, 5-LO – Lys-655, has been shown to be necessary for the redis- is activated, translocates to a membrane compartment, and tribution of 5-LO to the nuclear compartment [64,97]. gets rapidly inactivated, in a Ca2+-dependent manner Moreover, very recent data indicate that also the N- [159,162,197]. Experiments with mouse mast cells stimu- terminal β-barrel domain in 5-LO plays a role in this pro- lated with ionophore A23187 or anti-IgE, showed that cess [22]. In this context, it is interesting to note that a translocation may be reversible and that the strength and variant of 5-LO, post-translationally modified by phos- duration of the Ca2+ influx, may determine the reversibil- phorylation, has been identified [98]. The phosphorylated ity of translocation as well as the extent of enzyme inacti- 5-LO accumulated in the nuclear fraction of activated HL- vation [103]. Furthermore, using the reversible 5-LO

14

Introduction

60 cells, suggesting that also phosphorylation may be quence comparisons revealed that the receptor is distantly functionally coupled to the 5-LO translocation process. related to certain somatostatin receptors as well as some Further analysis of the enzyme compartmentalization of the chemokine receptors, e.g., those which bind fMLP, revealed that 5-LO can also be present in the nucleus of LXA4, and C5a [142,202]. Interestingly, the profound resting cells associated with the nuclear euchromatin, a chemotactic activity and dominant role of LTB4 in reper- site from which it translocates to the nuclear envelope fusion injury was demonstrated in a rat model of kidney [198]. In polymorphonuclear neutrophils, 5-LO transloca- ischemia, using CHO cells stably expressing the BLT1 tion was associated with functional responses such as receptor [134]. activation, adherence, and increased ability to synthesize Furthermore, the gene structure and promoter region LTB4 [18]. On the other hand, adherence of eosinophils of the BLT1 receptor has been characterized [81]. Thus, was accompanied by reduced capacity to synthesize LTC4 Sp1 was found to be a strong activator of transcription and [17]. This effect was explained by a resistance to activa- the CpG sites of the promoter were highly methylated in tion of the nuclear pool of 5-LO. Apparently, the subcellu- cells not expressing the BLT1 receptor, suggesting that lar distribution, directional migration, and activation of 5- DNA methylation is involved in the regulation of cell- LO in response to Ca2+ mobilization appear to differ specific receptor expression. between species and cell types [19]. As previously dis- The role of the BLT1 receptor has also been studied by cussed, cPLA2 also translocates to the nuclear membrane targeted gene disruption [62,181]. The receptor was nec- upon cell stimulation and LTC4 synthase seems to reside essary to elicit the physiological effects of LTB4 (e.g. at the same site [48,148,171]. In fact, LTA4 hydrolase is chemotaxis, calcium mobilization, and adhesion to endo- the only enzyme which is not membrane associated in thelium) and important for the recruitment of leukocytes resting cells, and which appears to stay in the cytosol in in an in vivo model of peritonitis. As previously observed activated cells (cf. Fig. 4). in mice lacking 5-LO, FLAP, or LTA4 hydrolase, BLT1 Together, these findings imply that leukotriene biosyn- (-/-) mice were protected from the lethal effects of PAF- thesis is executed by a complex of enzymes assembled at induced anaphylaxis. Interestingly, the protection was the nuclear membrane. This conclusion in turn suggests predominantly observed in female mice [62]. that these enzymes and/or their products may have addi- A second G protein-coupled 7TM receptor for LTB4, tional intracellular/intranuclear functions, perhaps related BLT2, has recently been identified [78,184,204]. This to signal transduction or gene regulation. In line with this receptor is homologous to the BLT1 receptor but has a notion, it has been reported that LTB4 is a natural ligand higher Kd value for LTB4 (23 nM) and a different ligand- to the nuclear orphan receptor PPARα, suggesting that specificity and binding profile for various BLT antago- LTB4 may have intranuclear functions possibly coupled to nists [203]. Thus, BLT2 binds and is activated by 12S- lipid homeostasis [31]. In a recent study, it was also re- HETE and 15S-HETE. Moreover, BLT2 is ubiquitously ported that 5-LO can interact with several cellular pro- expressed in various tissues, in contrast to the BLT1 re- teins, including coactosine-like protein (CLP) and trans- ceptor, which is predominantly found in leukocytes. The forming growth factor type β-receptor-I-associated protein genes encoding the LTB4 receptors are located on chro- (TRAP-1) [152]. In addition 5-LO interacts with a third mosome 14 [78,132]. Interestingly, the open reading protein which is a human homologue of the protein frame of the gene encoding the BLT2 receptor is located “Dicer”, a member of the RNase III family of nucleases, within the promoter region of the BLT1 receptor gene, an which is implicated in the RNA interference mechanism unusual gene structure previously not described among of gene regulation [8,152]. However, the biological sig- mammals, suggesting that the regulation of these two nificance of these protein-protein interactions is presently genes are linked together [81]. unclear. 1.9.2. Receptors for cys-LTs, CysLT1, and CysLT2 - 1.9. Leukotriene receptors. The cys-LTs are recognized by at least two receptor types The biological effects elicited by leukotrienes are medi- (CysLT1, and CysLT2), both of which have been cloned ated via specific cell surface receptors that for long es- and characterized as G protein-coupled 7TM receptors caped purification and cloning. However, a dramatic pro- [66,102,135,169,182]. The CysLT1 receptor contains 337 gress has recently occurred in this important research amino acid residues and mRNA is found in, e.g., the field, beginning with the cloning and characterization of spleen, peripheral blood leukocytes, lung tissue, smooth the major receptor for LTB4. muscle cells, and tissue macrophages [102,169]. The preferred ligands for the CysLT1 receptor are LTD4 fol- 1.9.1. Receptors for LTB4, BLT1, and BLT2 - For LTB4, lowed by LTC4 and LTE4 in decreasing order of potency. two types of surface receptors, with different affinity and Furthermore, the CysLT1 receptor-specific antagonists, ® ® cellular expression, are known (BLT1 and BLT2). The montelukast (Singulair ), zafirlukast (Accolate ), and ® BLT1-receptor has been cloned and characterized as a 43 (Onon ) are able to inhibit LTD4-induced kDa, G protein-coupled receptor with seven- Ca2+ mobilization in cells expressing the receptor. The transmembrane-spanning domains (7TM) [202]. The gene encoding the receptor is located on the X chromo- BLT1 receptor is only expressed in inflammatory cells some [102]. [81] and shows a high degree of specificity for LTB4 with The CysLT2 receptor contains 346 amino acids with a Kd of 0.15 -1 nM [202,204]. Computer assisted se- approximately 40% sequence identity to the CysLT1 re-

15 Martin J. Mueller ceptor [66,135,182]. This receptor binds LTC4 and LTD4 nickel-nitrilotriacetic acid resin, followed by FPLC on a equally well, whereas LTE4 and the antagonists men- hydroxyapatite column. tioned above show low affinities to the receptor. Studies Protein concentrations were determined by the Brad- on the tissue distribution of the CysLT2 receptor show ford method using bovine serum albumin as a standard high levels of mRNA in, e.g., heart, brain, peripheral [16], or by UV spectrophotometry using the equation: blood leukocytes, spleen, placenta, and lymph nodes, protein concentration = (A280 x 1.4 - A260 x 0.7) mg/ml. whereas only small amounts are found in the lung. The The purity of the enzymes was checked by SDS-PAGE functional role(s) of the CysLT2 receptor is presently which was carried out on Phast 10-15 % gradient gels unclear and its wide tissue distribution opens for many using a Pharmacia Phast system or in a Mini-protean II possibilities, including regulation of brain and/or cardiac dual slab cell according to Laemmli [91]. The protein functions. bands were visualized by staining with Coomassie bril- liant blue.

2. AIMS OF THE PRESENT INVESTIGATION 3.2. Site-directed mutagenesis. The human LTA4 hydrolase cDNA was mutated by using LTA4 hydrolase catalyzes the final step in the biosynthesis the so-called "megaprimer" method [170]. Site-directed of LTB4, a potent chemoattractant and immune- mutagenesis was carried out on the E. coli expression modulating lipid mediator. Due to its biological properties vector pT3-MB4, which contains the entire coding se- and likely involvement in inflammatory diseases, it was of quence for human LTA4 hydrolase cDNA and six addi- medical interest to study how this enzyme is regulated at tional histidine codons introduced immediately after the the molecular level. The general aim of the present thesis initiation codon. In the first step a megaprimer was ampli- was to carry out biochemical studies to identify structural fied, by using the internal primer A containing the desired and functional elements in LTA4 hydrolase and to exam- mutation, together with primer B, covering a unique re- ine the molecular events by which LTA4 binds to and striction site. The megaprimer and primer C were used in block the active site of the enzyme. This in turn would a second PCR reaction, to generate the mutated fragment. possibly generate information, which can be used in the After digestion of the 5’ and 3’ ends of the fragment with design of potent and specific enzyme inhibitors. The re- BglII and BfrI, it was ligated into the pT3-MB4 vector, search tasks of the present thesis may be summarized as opened with the same restriction enzymes. follows: In almost all PCR reactions we used the Pfu poly-

merase isolated from the marine archaebacterium Pyro- • Carry out studies with chemical modification of LTA 4 coccus furiosus, instead of the normally used Taq poly- hydrolase to identify which types of amino acid resi- merase. Since Pfu polymerase lacks the 3’ to 5’ exonucle- dues are present at the active site. ase activity reported for Taq polymerase, the ends of the • Characterize a peptide map for LTA hydrolase. 4 PCR fragments will remain intact and blunt, thus allowing • Use differential peptide mapping and Edman degrada- blunt-end ligation. The 3’ to 5’ proofreading activity asso- tion to identify segments of the protein that are cova- ciated with Pfu polymerase also results in a greatly in- lently modified by various chemical reagents. creased fidelity in DNA synthesis as compared with Taq polymerase [101]. • Apply differential peptide mapping and Edman degra- The entire mutated cDNAs were sequenced by the dation to identify a protein segment in LTA4 hydrolase dideoxy chain termination method to verify the mutations to which LTA binds during suicide inactivation. 4 and to confirm that no other changes in the nucleotide • Carry out site-directed mutagenesis of residues impli- sequence had occurred [168]. cated in suicide inactivation and analyze the molecular mechanism(s) involved in this process. 3.3. Activity determinations. 3.3.1. Epoxide hydrolase activity - Typically, the ep- oxide hydrolase activity was assayed by incubating ali- 3. METHODOLOGY quots of purified LTA4 hydrolase with 4-9 nmol LTA4 lithium salt. The incubations were performed in 100-200 3.1. Enzyme purification and expression of recombi- µl 50 mM Tris-Cl or Hepes buffer, pH 8, at room tem- nant enzymes. perature. After 15 s the reactions were quenched with 2 Human leukocyte LTA4 hydrolase and human recombi- vol. methanol containing PGB1 as the internal standard. nant enzyme were purified to apparent homogeneity by The samples were acidified to pH 3 and subjected to precipitation with streptomycin sulfate and ammonium solid-phase extraction with Chromabond C18 columns. sulfate followed by fast protein liquid chromatography The product formation was quantitated by reverse phase- (FPLC) on anion exchange (Mono-Q), hydrophobic inter- HPLC, based on peak area integrations normalized with action (Phenyl-Superose), and chromatofocusing (Mono- respect to the internal standard. P) columns. Alternatively, the human recombinant enzymes were 3.3.2. Peptidase activity - The aminopeptidase activity expressed as (His)6-tagged fusion proteins in E. coli was determined spectrophotometrically in 250 µl 50 mM (JM101) and purified by affinity chromatography on a Tris-Cl buffer, pH 7.5, containing 100 mM NaCl with

16

Results alanine-4-nitroanilide as the substrate. The assays were Packard, model 5970B mass selective detector, connected performed in the wells of a microtiter plate at room tem- to a Hewlett-Packard, model 5890 gas chromatograph perature and the product formation (4-nitroaniline) was equipped with a methyl silicone capillary column (length measured as an increase in the absorbance at 405 nm 12 m, film thickness 0.33 µm). The carrier gas was helium using a Multiscan 96-well spectrophotometer. Quantita- at a flow rate of 38 cm/s. Injections were made in the split tions were made from a standard curve obtained with mode at an injector temperature of 200ºC. The initial known amounts of 4-nitroaniline. column temperature was 120ºC and was raised at 10ºC/min until 240ºC. For GC/MS, samples were con- 3.4. Chemical modification. verted to the methyl ester, trimethylsilyl ether derivatives, Aliquots of purified enzyme (5 µg in 100 µl of buffer) by treatment with diazomethane in diethyl ether followed were treated with the respective modifying reagent dis- by a mixture of hexamethyldisilazane and trimethylchlo- solved in an appropriate solvent. After 15 – 120 min of rosilane in pyridine. incubation, the reactions were terminated by gel filtration followed by enzyme activity determinations. Protection 3.7.3. Ultraviolet (UV) spectrophotometry - Ultravio- from inactivation was carried out by preincubating the let (UV) spectra were recorded on a Hewlett-Packard enzyme with either bestatin (2 mM) or captopril (10 mM) 8450 A Spectrophotometer with methanol as solvent. for 45 min at room temperature before the addition of modifying reagent. 3.7.4. Circular dichroism spectrometry (CD) - Circu- lar dichroism spectrometry of purified recombinant en- 3.5. Preparation of suicide inactivated enzyme. zymes was performed in 10 mM sodium phosphate buffer, Typically, 200 µg of LTA4 hydrolase (wild type and mu- pH 7.5, with an AVIV circular dichroism spectrometer tants) in 2 ml of buffer was incubated with LTA4, LTA4 model 62DS. methyl ester or LTA4 ethyl ester (14 µM) for 30 min at room temperature. The addition of leukotriene epoxides was repeated four times resulting in a final concentration 4. RESULTS of 60-70 µM. After the final addition, an aliquot of the enzyme sample was gel-filtered prior to determinations of 4.1. LTA4 hydrolase contains essential tyrosyl and residual epoxide hydrolase and peptidase activities. The arginyl residues at the active site (Paper I). remaining portion was subjected to proteolytic digestion We used chemical modification to identify residues essen- with Lys-C protease followed by peptide mapping. tial for the two catalytic activities of LTA4 hydrolase. Both enzyme activities were rapidly inactivated with N- 3.6. Peptide mapping. acetylimidazole and tetranitromethane, reagents fairly For peptide mapping, the enzyme preparations were dried specific for Tyr residues. Furthermore, treatment with 2,3- under N2 and denatured in 6 M guanidinium chloride / 0.4 butanedione and phenylglyoxal, regarded as specific for M Tris-Cl / 2 mM EDTA, pH 8.15, followed by reduction Arg residues, also resulted in loss of both activities. LTA4 with dithiothreitol for 30 min at 38oC. After carboxy- hydrolase could be protected from inactivation by these methylation with 100 mM sodium iodoacetic acid the tyrosyl and arginyl reagents by the competitive inhibitors, enzymes were cleaved overnight with Lys-C protease in bestatin and captopril, respectively. Using differential 1.5 M deionized urea / 50 mM borate buffer, pH 8. The labeling techniques we could also calculate that two Tyr peptides formed were separated by reverse-phase HPLC and three Arg were protected by the inhibitors, suggesting on a C18 column using a linear gradient of acetonitrile in that these residues are located close to or at the active 0.1 % trifluoroacetic acid. The absorbance was monitored center of the enzyme. at 214 and 280 nm for detection of both peptide bonds Limited modification of LTA4 hydrolase by thiol re- and aromatic amino acids, respectively. agents, in particular methyl methanethiosulfonate (MMTS), led to a >10-fold increase in the peptidase activ- 3.7. Instrumental analysis. ity and from a kinetic analysis we could conclude that this 3.7.1. Reverse-phase high performance liquid chro- was due to an increased turnover without significant al- matography (RP-HPLC) - For reverse-phase (RP)-HPLC teration of the Michaelis constant. The modified enzyme a column packed with Nova-Pak C18 (Radial Pak car- also displayed a reduced apparent affinity constant for tridge, 4 µm, 5 mm x 100 mm, Waters) was eluted with a chloride ions, which are known to stimulate the peptidase mixture of methanol:water:acetic acid (70:30:0.01) or activity. The effects of MMTS could not be prevented by acetonitrile:methanol:water:acetic acid (30:35:35:0.01) at incubation with competitive inhibitors suggesting that they a flow rate of 1.2 ml/min. The absorbance of the eluate do not result from modification of an active site residue. was monitored at 270 nm. Quantifications of the product were made by area integration based on a standard curve 4.2. Identification of a central peptide segment in obtained from analysis of known amounts of the internal LTA4 hydrolase to which LTA4 binds during suicide standard PGB1 and of the compound to be quantitated. inactivation (Paper II). LTA4 hydrolase is irreversibly inactivated and covalently 3.7.2. Gas chromatography / mass spectrometry modified when exposed to its lipid substrate LTA4. We (GC/MS) - Gas chromatography linked to mass spec- used differential lysine-specific peptide mapping of un- trometry (GC/MS) was performed with a Hewlett- modified and suicide inactivated LTA4 hydrolase to iden-

17 Martin J. Mueller

O COOH

LTA4 70-80% 20-30%

OH OH OH OH

COOH COOH

LTB ∆∆68----LTBtrans cis 4 4

6 8 Fig. 5. Formation of LTB4 and ∆ -trans-∆ -cis-LTB4 by mutants at position 378.

tify a 21-residue peptide (termed K21), encompassing the 4.4. Mutants of Tyr-378 can hydrolyze LTA4 into a amino acid residues 365-385 of human LTA4 hydrolase, double-bond isomer of LTB4 (Paper IV). which is involved in the binding of LTA4, LTA4 methyl When the catalytic properties of [Y378F] and ester, and LTA4 ethyl ester to the native protein. Amino [Y378Q]LTA4 hydrolase were studied in detail we found acid sequence analysis of a modified form of this peptide, that both mutants could convert LTA4, not only into generated by treatment with LTA4 ethyl ester, revealed LTB4, but also into a second, previously unidentified, that modification had occurred at Tyr-378 in LTA4 hy- metabolite. This metabolite was structurally identified by drolase. The degree of peptide modification corresponded RP-HPLC, UV-spectrometry, GC-MS, UV-induced dou- well to the degree of enzyme inactivation. Furthermore, ble-bond isomerization, and cochromatography with syn- both inactivation and covalent modification could be thetic standards as 5S,12R-dihydroxy-6,10-trans-8,14-cis- 6 8 prevented by preincubation with the competitive inhibitor eicosatetraenoic acid, i.e., ∆ -trans-∆ -cis-LTB4 (Fig. 5). bestatin. The relative formation of this compound versus LTB4 by [Y378F] and [Y378Q]LTA4 hydrolase was 18% and 32%, 4.3. LTA4 hydrolase can be protected from suicide respectively. inactivation by mutation of Tyr-378 (Paper III). To detail the role of Tyr-378 for inactivation/covalent 4.5. Evidence that the substrate-mediated inactivation modification of LTA4 hydrolase by LTA4, this residue of LTA4 hydrolase follows an affinity-labeling mecha- was exchanged for a Phe or Gln. In addition, two adjacent nism (Paper V). and potentially reactive Ser residues (Ser-379 and Ser- Previous investigators have found that substrate-mediated 380), were each exchanged for Ala in two separate mu- inactivation/covalent modification of LTA4 hydrolase tants. The mutated enzymes were expressed as (His)6- satisfies criteria defining a mechanism-based process, e.g., tagged fusion proteins in E. coli, purified to apparent the epoxide hydrolase and peptidase activity are lost si- homogeneity and characterized. multaneously and irreversibly in a time-dependent, satur- When the mutated enzymes were subjected to repeated able process with active site-specificity and a 1:1 exposures of LTA4, followed by peptide mapping and stoichiometry between lipid and protein [138,140]. Given enzyme activity determinations, we found that wild type a mechanism-based process, the inactivation would be enzyme, [S379A], and [S380A]LTA4 hydrolase were dependent on turnover and vice versa. To test whether this equally susceptible to suicide inactivation. In contrast, the is actually the case for LTA4 hydrolase, we investigated two mutants in position 378 were no longer inactivated or the interaction of the catalytically incompetent covalently modified by LTA4. Moreover, the most con- [E318A]LTA4 hydrolase with LTA4 and LTA4 methyl servative replacement at position 378, i.e., [Y378F]LTA4 and ethyl ester. We also investigated the mutated enzymes hydrolase, exhibited an increased (2.5-fold) turnover of [Y378F/Q]LTA4 hydrolase that are resistant towards LTA4 as compared to the wild type enzyme. Hence, a substrate-mediated inactivation. After exposure to the single amino acid exchange at position 378, generates a epoxides, the proteins were subjected to differential pep- recombinant enzyme with an increased catalytic efficiency tide mapping and enzyme activity determinations. and resistance to suicide inactivation. Peptide maps of untreated [E318A]LTA4 hydrolase and [E318A] exposed to LTA4 (5 x 14 µM), were identi-

18

Discussion cal except for peptide K21, previously identified as the peptide to which LTA4 binds during suicide inactivation (Paper II and III). Thus, in peptide maps of enzyme treated with LTA4, K21 was reduced to approximately Captopril 25% of the untreated control. For the catalytically active wild type enzyme, the modification as well as the corre- Bestatin sponding inactivation by LTA4 did not exceed 50 % of the A r g g r untreated control. Under the same conditions, LTA4 A methyl or ethyl ester reacted with [E318A]LTA hy- T 4 r y r y drolase in a similar way, leading to a specific modification T of peptide K21 as judged by its disappearance (remaining Arg peptide 18 % and 10 % of the control, respectively) in the corresponding peptide maps. The extent of peptide modi- fication when [E318A]LTA4 hydrolase was treated with LTA4 methyl and ethyl ester, was comparable to the cor- Enzyme responding modification of peptide K21 using wild type enzyme (23 % and 14 % of the control, respectively). Fig. 6. Two tyrosines and three arginines at, or close to, the active site of LTA4 hydrolase.

5. DISCUSSION

any of the 11 Cys residues in the catalytic mechanism is 5.1. Identification of functional Tyr and Arg residues unlikely. Interestingly, some of the thiol reagents and at the active site of LTA hydrolase. 4 particularly MMTS increased the peptidase activity at the Chemical modification is a useful method for screening of beginning of the modification process. The increased amino acid residues, which are essential for substrate peptidase activity after short time incubation with MMTS binding and catalysis. It is particularly useful to have was shown to be due to a drastically increased enzyme competitive inhibitors for the enzyme, since protection turnover (k ) whilst Michaelis constant (K ) was un- against inactivation by such inhibitors is the usual crite- cat m changed, indicating that the binding of the substrate rion to assess that modification is active site-directed. alanine p-nitroanilide was not affected. Chloride and sev- Exposure of LTA hydrolase to the tyrosine reagent N- 4 eral other monovalent anions are known to stimulate the acetylimidazole resulted in a rapid inactivation of both the peptidase activity by increasing V without affecting K peptidase and the epoxide hydrolase activity. Notably, max m [193] and it could be demonstrated that MMTS-modified addition of hydroxylamine could restore both catalytic enzyme displayed a higher affinity for chloride ions. In activities which strongly indicates that the inactivation addition, MMTS-modified enzyme displayed a higher was due to modification of tyrosine residue(s). Further- peptidase activity even in the absence of chloride ions. more, the competitive inhibitor bestatin could partially Thus, certain thiol reagents may induce small changes in protect the enzyme from N-acetylimidazole, suggesting the structure leading to a conformation with an increased that critical tyrosine(s) are located close to, or at, the ac- affinity for stimulatory chloride ions and a higher catalytic tive site(s). Similar results were obtained with another efficiency. Prolonged, more extensive thiol modification tyrosine reagent, i.e. tetranitromethane, and from meas- inactivated slowly the peptidase as well as the epoxide urements of nitrated residues by UV, in the presence or hydrolase activity, perhaps due to more drastic changes of absence of bestatin, it was calculated that two out of 22 the tertiary structure. tyrosines could be protected by the competitive inhibitor which would thus correspond to the number of critical 5.2. Identification of molecular determinants for sui- tyrosines present at the active site (Fig. 6). cide inactivation of LTA hydrolase. Two arginine-modifying reagents, viz. 2,3-butanedione 4 Typically, LTA hydrolase is covalently modified and and phenylglyoxal, effectively inhibited both enzyme 4 inactivated by its endogenous lipid substrate LTA , a activities. As was the case for N-acetylimidazole and 4 process referred to as suicide inactivation [38,115]. This tetranitromethane, the enzyme could be protected from built-in mechanism for enzyme inhibition may be of im- inactivation by competitive inhibitors, i.e., either bestatin portance for the overall regulation of LTB biosynthesis or captopril. The number of arginines involved in the 4 in vivo. Using enzyme kinetics, Fitzpatrick and coworkers inactivation process were determined from the incorpora- have shown that suicide inactivation satisfies several crite- tion of [7-14C]phenylglyoxal into unprotected and pro- ria which define a mechanism-based process [138,140]. tected enzyme. Thus, using this technique with differential For instance, after treatment of LTA hydrolase with labeling, it was calculated that three residues out of a total 4 LTA or with LTA methyl ester, the epoxide hydrolase of 23 Arg present in LTA hydrolase, could be protected 4 4 4 and peptidase activities are lost simultaneously and irre- by captopril (Fig. 6). versibly in a time-dependent, saturable process that is of Since inactivation by highly reactive and specific thiol pseudo first-order kinetics and dependent upon catalysis. reagents was poor and could not be prevented by preincu- Active site-specificity has been demonstrated by protec- bation with a competitive inhibitor, the involvement of

19 Martin J. Mueller

Peptide K21

365378 383 385

L V V D L T D I D P D V A YY S S V P G K

1 45 610 NH COOH 2

V I A HH E I S S W T-(X)11 -F W L N E G H T V 295 299 318 296

Zinc binding motif

Fig. 7. Two catalytic domains in LTA4 hydrolase. tion with competitive inhibitors and mass spectrometric the N-terminal amino acid sequence, which was in agree- analysis has revealed that suicide inactivation occurs pre- ment with the corresponding sequence of K21. Hence, dominantly in a 1:1 stoichiometry between lipid and pro- suicide inactivation of LTA4 hydrolase is accompanied by tein, with only little modification of secondary sites. a shift of peptide K21 to the lipophilic region of the pep- tide map where it appears as K21-LT. 5.2.1. Identification of a peptide region involved in LTA4 hydrolase can also be suicide inactivated by suicide inactivation - To identify molecular determinants LTA4 methyl and ethyl ester. In these cases, the inactiva- of suicide inactivation, we turned to differential lysine- tion is more efficient, which may be explained by less specific peptide mapping. Thus, LTA4 hydrolase was depletion of the leukotriene epoxides due to the absence treated with repetitive additions of LTA4 to obtain sui- of significant enzymatic conversion and a higher chemical cide-inactivated enzyme followed by digestion with Lys-C stability of esterified LTA4 as compared to the free acid. protease. The resulting peptides were separated by RP- We also tested these LTA4 analogs in experiments similar HPLC to obtain a peptide map. In parallel experiments, to those described above. Again, we found loss of peptide identical peptide digests were prepared from suicide- K21 in digests of inactivated enzyme and also observed inactivated and from unmodified enzyme for differential the simultaneous appearance of modified, more lipophilic analysis by HPLC. One peak was always reduced in the peptides. In the case of LTA4 ethyl ester, the amounts of peptide map of suicide inactivated enzyme. This peptide modified peptide, denoted K21-LTet, was sufficient to peak was collected and subjected to amino acid sequence allow complete Edman degradation. Thus, the sequence of analysis which revealed a peptide spanning 21 residues K21-LTet was identical to the sequence of K21 with the from Leu-365 to Lys-385. Due to the number of amino exception of a gap in position 14, corresponding to Tyr- acids and the fact that it originates from a Lys-C digest, 378 of intact LTA4 hydrolase. Apparently, LTA4 ethyl the peptide was denoted K21 (Fig. 7). ester had bound to Tyr-378 during suicide inactivation of To link peptide K21 to suicide inactivation, the loss of LTA4 hydrolase. enzyme activities upon repetitive additions of LTA4 was Suicide inactivation and covalent modification (as compared with the loss of K21 in corresponding peptide judged by loss of peptide K21 in peptide maps) by the maps. There was a good correlation between these two leukotriene epoxides could be prevented by preincubation parameters which indicated that the heneicosapeptide is of the enzyme with the competitive inhibitor bestatin. indeed involved in the covalent binding of the substrate Hence, binding of LTA4 and its esters to peptide K21 is LTA4 to the protein. Further evidence for this conclusion an active site-directed process and K21 most likely con- was obtained by the identification of a modified peptide tains one or several active site residues. In fact, peptide K21, denoted K21-LT (to indicate the content of a leukot- K21 could be considered as a second catalytic domain, in riene moiety), which eluted in the more lipophilic part of addition to the previously well-characterized zinc-binding the HPLC chromatogram. Although the recovery of K21- site (Fig. 7). Although the borders of K21 are defined by LT was poor, enough material could be collected to trace

20

Discussion

Fig. 8. Nonenzymatic and enzymatic hydrolysis of LTA4.

6 8 the proteolytic specificity of Lys-C protease, this region convert LTA4 into both LTB4 and ∆ -trans-∆ -cis-LTB4, would seem to be functionally centered around the two just like the human mutant of Tyr-378 [87,180]. The yeast tyrosines in position 378 and 383. This conclusion has enzyme also displays a reduced sensitivity to suicide inac- recently been corroborated by X-ray crystallography (see tivation by LTA4. This in turn suggests that during evolu- section 5.5.3.). tion, the enzyme’s product specificity has improved in favor of LTB4. At the same time, LTA4 hydrolase has 5.2.2. Tyrosine-378 is a structural determinant for been penalized with an increased catalytic restrain im- suicide inactivation - To investigate the role of Tyr-378 in posed by covalent modification/inactivation. suicide inactivation and its potential catalytic function we carried out a mutational analysis. Thus, we exchanged Tyr 5.2.4. Possible role of Tyr-378 in the hydrolysis of for Phe or Gln in two separate mutants. Interestingly, LTA4 into LTB4 - Leukotriene A4 is a highly unstable enzyme activity determinations of the purified proteins allylic epoxide which is spontaneously hydrolyzed in and differential peptide mapping (presence of peptide water with a t½ ≈ 10 s at neutral pH [41]. Non-enzymatic K21), before and after repeated exposure to LTA4, re- hydrolysis of LTA4 is thought to be initiated via an acid- vealed that the mutants in position 378 were no longer induced opening of the epoxide moiety and a carbocation, inactivated or covalently modified by LTA4. Furthermore, with a positive charge delocalized over the triene system, in [Y378F]LTA4 hydrolase, the value of kcat for epoxide would be formed as an intermediate in the reaction [13]. hydrolysis was increased 2.5-fold over that of the wild This intermediate will result in a planar sp2-hybridized type enzyme. Thus, by a single point mutation in LTA4 configuration at C-12, which in turn allows a nucleophilic hydrolase, catalysis and covalent modification/inactivation attack from both sides of the carbon. Accordingly, the two can be dissociated, yielding an enzyme with increased epimers at C-12 of 5(S),12-dihydroxy-6,8,10-trans-14-cis- 6 turnover and resistance to mechanism-based inactivation. eicosatetraenoic acid, also referred to as ∆ -trans-LTB4 6 and ∆ -trans-12-epi-LTB4, will be formed and are in fact 5.2.3. Formation of a double-bond isomer of LTB4 by the predominant non-enzymatic hydrolysis products of mutants of tyrosine 378 - A more detailed examination of LTA4 (Fig. 8). The structure of LTB4, formed by enzy- the catalytic properties of [Y378F], [Y378Q], [S379A], matic hydrolysis, differs from the structure of either of the and [S380A]LTA4 hydrolase revealed that the two mu- two non-enzymatically formed 5,12-dihydroxy acids in tants in position 378 were able to generate not only LTB4 two ways, viz. the double-bond geometry and the configu- but also a second metabolite of LTA4, in a yield of about ration of the hydroxyl group at C-12. Apparently, during 20-30% [IV]. From data obtained by UV spectrophotome- enzymatic hydrolysis of LTA4 into LTB4, LTA4 hy- try, GC/MS, UV-induced double-bond isomerization, and drolase ensures a stereoselective introduction of H2O at comparison with a synthetic standard, the novel metabo- C-12 as well as the formation of the thermodynamically lite was assigned the tentative structure 5(S),12(R)- less favored ∆6-cis-∆8-trans-∆10-trans configuration of dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, i.e., the conjugated triene (Fig. 8). Interestingly, the mutants at 6 8 ∆ -trans-∆ -cis-LTB4 (Fig. 5). The observation that mu- position 378 differ from wild type enzyme regarding one tants in position 378 can generate an isomer of LTB4 of these two essential functions of the enzyme, i.e., the certainly corroborated our previous conclusion that Tyr- positioning of the cis double bond in the product. Hence, 378 is an active site residue and suggested that the residue Tyr-378 appears to be involved in this aspect of catalysis, participates in epoxide hydrolase catalysis. perhaps by assisting in the proper alignment of LTA4 in Only two non-mammalian LTA4 hydrolases have been the substrate-binding pocket or by promoting a favorable characterized to date, viz., enzyme from X. laevis and S. conformation of a putative carbocation intermediate. cerevisiae. In both cases, the purified proteins are able to

21 Martin J. Mueller

5.3. Molecular mechanism of substrate-mediated inac- tivation of LTA hydrolase. A Mechanism-based inactivation 4 k A mechanism-based inhibitor is an unreactive molecule 3 LTB4 + E which is activated by the catalytic machinery of an en- k1 k2 zyme into a reactive product which in turn will covalently LTA4 + E LTA4. E LT * . E k k modify an active site residue (Fig. 9). Typically, a mecha- -1 -2 k4 LT - E nism-based inhibitor is a synthetically prepared substrate analogue that has been designed to covalently bind a cata- lytic acid-base of the transition state after its enzymatic B Affinity labeling transformation into a reactive species [39,89]. Since the k mechanism-based inhibitor requires the catalytic appara- 2 LTB4 + E k1 tus for its action, this type of compound is ideal for the LTA4 + E LTA4. E development of specific enzyme inhibitors. k-1

An affinity label, on the other hand, is a molecule k3 LT - E which is chemically reactive in itself and therefore does not need enzymatic processing to become destructive (Fig. 9). This type of inhibitor, which resembles the substrate, Fig. 9. Schematic representation of mechanism-based will also modify the active site because of its binding inactivation (A) and affinity labeling (B). specificity. An affinity-label could potentially also react with any protein to which it binds with sufficient strength. Thus, the distinction between a mechanism-based inhibi- tor and an affinity label has important pharmacological an affinity labeling mechanism [89] in which LTA4 may and biological implications. simply become captured by a proximate nucleophilic A critical point in deciding which mechanism best residue at the active site, namely Tyr-378. describes the substrate mediated inactivation of LTA4 hydrolase will be to determine whether or not the enzyme 5.3.3. LTA4 act as an affinity label during self- machinery is required for generation of the reactive inac- catalyzed inactivation of LTA4 hydrolase - Based on pri- tivating molecule. One possible species could be a carbo- marily kinetic data, suicide inactivation of LTA4 hy- cation which is formed during non-enzymatic hydrolysis drolase has been interpreted as a mechanism-based proc- of LTA4 and which also seems to be an intermediate in ess. However, this model is difficult to reconcile with the conversion of LTA4 into LTB4 [10]. certain properties of LTA4. For instance, the molecule LTA4, its ester derivatives and structurally related allylic 5.3.1. The catalytically inactive mutant [E318A]LTA4 epoxides are chemically labile and react indiscriminately hydrolase is covalently modified by LTA4 - Mutagenetic with all nucleophiles available (t½ < less than 10 s in replacement of any of the three zinc binding ligands in aqueous solutions) and an additional step of substrate LTA4-hydrolase, including Glu-318, leads to the com- activation does not seem to be required for covalent bond bined loss of zinc and enzyme activities [116]. The mutant formation/inactivation. The importance of the chemical [E318A]LTA4 hydrolase was selected to study the effects reactivity of the allylic epoxide is also demonstrated by of leukotriene epoxides on an enzyme lacking a key ele- the fact that other structurally dissimilar allylic epoxides ment of catalysis, the fundamental basis for a mechanism- [136], also inactivate LTA4 hydrolase whereas non-allylic based process. Exposure of [E318A]LTA4 hydrolase to epoxides do not. LTA4 or LTA4 methyl or ethyl ester resulted in covalent As one would expect from an affinity label, LTA4 has modification of peptide K21 to the same extent as for wild been shown to inactivate enzymes other than LTA4 hy- type enzyme. This finding is difficult to rationalize within drolase, e.g., 5-LO [95]. The catalytic apparatus of a li- the concept of a mechanism-based process in which cata- poxygenase would be expected to be greatly different lytic activation of the substrate is necessary and precedes from that of an epoxide hydrolase. However, binding covalent modification and inactivation. In fact, the degree specificity of LTA4 to lipoxygenases is reasonable, given of modification was even higher in the absence of cataly- the structural similarity between LTA4 and arachidonic sis. acid, a natural substrate of lipoxygenases. Accordingly, the Ki of LTA4 versus 5-LO was calculated to approx. 2 5.3.2. Covalent modification of a non-essential resi- µM. Hence, the affinity between LTA4 and 5-LO appears due at the active site of LTA4 hydrolase - Apparently, to be of the same, or even higher, strength as that reported Tyr-378 is as a major site to which LTA4 binds during for LTA4 hydrolase (Km in the range of 5-30 µM). suicide inactivation (Paper III). Exchange of Tyr-378 for a Taken together, we think that this large body of data is Phe or Gln rendered the enzyme virtually resistant to best explained by LTA4 acting as an affinity label. In this suicide inactivation. In addition, [Y378F]LTA4 hydrolase model, LTA4 is the natural substrate of the enzyme and at exhibited an increased kcat for LTA4. Hence, turnover was the same time, by virtue of its chemical reactivity, an completely dissociated from inactivation which shows that efficient affinity label. After initial binding of LTA4 to the Tyr-378 is not critical for catalysis. Modification of a active site, the hydroxyl group of Tyr-378 may attack the nonessential active site residue would be consistent with reactive allylic epoxide, or a spontaneously formed carbo-

22

Discussion cation thereof, to cause covalent modification/enzyme tion is poor. Moreover, it has been suggested that radical inactivation. Moreover, this mechanistic model agrees and carbocation transition-state intermediates or malon- well with the current knowledge about LTA4 hydrolase, dialdehyde formed during catalysis participate in the the kinetics of catalysis and inactivation, the chemistry of chemical modifications underlying inactivation of PGI LTA4, and the properties of the mutated enzymes. An synthase [65,187]. affinity labeling mechanism predicts (i) that LTA4 is likely to destroy other enzymes/protein in its neighbor- 5.4.3. 5-Lipoxygenase - If one considers enzymes hood to which it binds with sufficient strength and (ii) that functionally coupled to LTA4 hydrolase, the upstream 5- LTA4 can not be used as a template for design of a sui- LO is another example of an unstable enzyme, which is cide-type inhibitors. susceptible to self-catalyzed inactivation. However, for this enzyme, the irreversible inactivation observed in 5.4. Suicide inactivation of other enzymes in the eico- whole cells is a complex process associated to several sanoid cascade. events including turnover-independent oxidation, mem- 5.4.1. Cyclooxygenases - Turnover dependent or self- brane association, and catalytic turnover of substrate. catalyzed suicide inactivation is a typical trait of many Exposure of recombinant 5-LO to oxygen and DTT enzymes involved in the biosynthesis of eicosanoids. For results in turnover-independent inactivation, apparently instance, COX-1 and COX-2 are highly unstable enzymes caused by oxidative damage by H2O2 and/or an activated that rapidly become inactivated during catalysis [178]. oxygen species formed via a Fenton type reaction of the 2+ Although the physiological significance of this process is Fe enzyme with H2O2 [150]. Since oxidatively inacti- not clear, the self-catalyzed inactivation and rate of syn- vated recombinant 5-LO was found to lack its iron con- thesis of new COX has been suggested to set a ceiling on tent, it has been suggested that the iron ligands are dam- a tissue’s ability to synthesize PGs in a given period of aged in this process [149]. It seems likely that radicals and time [110]. Cyclooxygenases possess two catalytic activi- activated oxygen species, released during catalysis, con- ties, the cyclooxygenase and peroxidase activity, each of tributes to the self-catalyzed inactivation as well (see which is inactivated by mechanism-based, first-order below). processes [178]. In addition, the enzyme is covalently When 5-LO becomes activated and translocates to the modified by arachidonate, which can bind to the protein. nuclear envelope, the resulting membrane association Although the molecular details of the inactivation process appears to inactivate the enzyme [159,162,197]. Since are unknown, it is regarded as an oxidative damage, par- these to events were shown to vary depending on the ticularly of the prosthetic heme, caused by the formation magnitude of the cellular stimulus used [103] and a com- of free radicals generated during the cyclooxygenase and petitive inhibitor afforded protection against inactivation peroxidase reactions [88,94]. Spectroscopic studies have [67], it was concluded that enzyme translocation / inacti- indicated that the self-catalyzed inactivation originates vation involves catalytic turnover. Interestingly, several from a reaction intermediate which does not, however, lines of evidence including studies of the inactivation of directly involve an arachidonate derived radical since 5-LO in phosphatidyl choline vesicles, have indicated that covalent binding of the fatty acid is slower than the loss of membrane association of the enzyme in fact promotes enzyme activity [88,94,200]. Furthermore, the inactivation turnover-independent oxidative damage in an active site- appears to involve the formation of a tyrosyl radical at a directed manner [27,80]. site different from the catalytic Tyr-385 [178,185]. How- Considering the self-catalyzed, turnover-dependent, ever, the nature of the protein modifications underlying inactivation of 5-LO, it is generally believed to be the COX self-inactivation have not yet been defined. Hence, result of protein damage caused by radical species gener- the inactivation of COX does not seem to be a classical ated by homolytic cleavage of a hydroperoxy intermediate mechanism-based, suicide inactivation in which an acti- by the peroxidase activity intrinsic to lipoxygenases. 5- vated substrate derivative destroys the enzyme. Lipoxygenase is a bifunctional enzyme catalyzing both a lipoxygenase and an LTA4 synthase reaction, sequentially 5.4.2. PGI synthase and TX synthase - Downstream of from arachidonic acid. The product of each of these reac- COX-1 and COX-2, are several specific synthases that tions could potentially contribute to the inactivation of 5- catalyze formation of PGE2, PGD2, PGF2α, PGI2 and LO. Thus, 5-HPETE has been shown to inactivate 5-LO, TXA2. The latter two enzymes, PGI synthase and TX perhaps via a radical mediated process [2]. In addition, synthase, both of which carry out a highly specific chem- kinetic studies have suggested that LTA4 can act as a istry, have been shown to undergo irreversible inactivation mechanism-based inhibitor, potentially via covalent cou- during catalysis [32,59,175]. Further kinetic characteriza- pling to the protein in a manner similar to what occurs in tions have indicated that these processes conform to a LTA4 hydrolase [96]. Covalent modification of 5-LO by mechanism-based, suicide type of inactivation [76,187]. some derivative of arachidonic acid has indeed been dem- For TX synthase it has been suggested that the inactiva- onstrated but the stoichiometry of the adduct or site(s) of tion mechanism involves a tight, non-destructive, associa- attachment between lipid and protein have not been de- tion of substrate or product with the prosthetic heme termined [96]. Moreover, oxidation of specific amino group [77]. On the other hand, spectroscopic studies have acids has not been demonstrated for 5-LO. Thus, a causal indicated that the heme in PGI synthase is altered or lost relationship between covalent modification and inactiva- [32], although the temporal correlation with the inactiva- tion remains to be established.

23 Martin J. Mueller

Fig. 10. Active site structure of human LTA4 hydrolase, including the zinc-binding ligands (His-295, His-299, and Glu- 318), Glu-296, Tyr-378, and Tyr-38. Adopted from [183].

5.4.4. 12- and 15-lipoxygenase - In analogy with 5- the most thoroughly studied and best understood example LO, porcine leukocyte 12-LO is inactivated by 15- of self-catalyzed suicide inactivation among members of HPETE, which is incorporated into the protein [84]. It has this enzyme family. been suggested that the inactivation of this lipoxygenase is caused by covalent binding of the second product, 14,15- LTA4, which is a highly reactive allylic epoxide, just like 5.5. Comparison of structure-function data generated LTA4 [84]. In this context it is interesting to note that with biochemical techniques or X-ray crystallography. inactivation of 15-LO was previously attributed to the Recently, the X-ray crystal structure of LTA4 hydrolase in oxidation of a single methionine into a methionine sulfox- complex with the competitive inhibitor bestatin was de- ide, by 13S-hydroperoxy-octadecadienoic acid (13- termined at 1.95 Å resolution [183]. The protein molecule HPODE) at the active site [63,154]. Later studies using is folded into an N-terminal, a catalytic, and a C-terminal differential peptide mapping identified Met-590 in human domain, packed in a flat triangular arrangement with ap- 15-LO as the residue oxidized by 13-HPODE [45]. Sur- proximate dimensions 85 x 65 x 50 Å3. Although the three prisingly, mutation of Met-590 into a Leu did not alter the domains pack closely and make contact with each other, a catalytic efficiency of the enzyme and did not protect it deep cleft is created between them. Interestingly, the cata- from inactivation by 13-HPODE [45]. Similar results have lytic domain is structurally very similar to the bacterial been obtained with porcine leukocyte 12-LO, which is protease thermolysin although the sequence identity is inactivated by eicosatetraynoic acid (ETYA) irrespective only about 7% over the corresponding polypeptide chains. of the presence of any of three methionines presumed to play a role in the loss of enzyme activity [155]. 5.5.1. The zinc site and catalytic residues - At the bot- In spite of intense research efforts, the mechanism(s) tom of the interdomain cleft, the zinc site is located. As and exact nature of the chemical modifications involved in predicted from previous work [116,195], the metal is self-catalyzed inactivation of central enzymes in the eico- bound to the three amino acid ligands, His-295, His-299, sanoid cascade, are not known. In some cases, however, and Glu-318 (Fig. 10). In the structure, however, Zn2+ is available data conform to a mechanism-based, suicide also bound to bestatin creating a pentavalent coordination. type process, involving several destructive intermediates In the vicinity of the prosthetic zinc, the catalytic residues and/or products, although the molecular details of the Glu-296 and Tyr-383 are located at positions that are reaction(s) are unclear. Other data seem to be better ex- commensurate with their proposed roles as general base plained by an affinity labeling mechanism. Thus far, sub- and proton donor in the aminopeptidase reaction. strate mediated inactivation of LTA4 hydrolase is clearly

24

Discussion

299 295 His His Glu318 2+ Zn H+ O- O +BH H H - O O A-

Fig. 11. Proposed reaction mechanism for enzymatic hydrolysis of LTA4 into LTB4. The shaded area indicates the L-shaped hydrophobic cavity in LTA4 hydrolase [183].

5.5.2. A binding site for LTA4 and its implications for entire modeled LTA4 molecule would then adopt a bent the epoxide hydrolase mechanism - Behind the pocket shape that fits very well with the architecture of the bind- occupied by the phenyl ring of bestatin there is an L- ing pocket. Hence, the critical double bond geometry at 6 shaped hydrophobic cavity approximately 6–7 Å wide, ∆ in LTB4 seems to be controlled by the exact binding which stretches 15 Å deeper into the protein (Fig. 11). conformation of LTA4 at the active site. Most of the residues lining the pocket are conserved and belong to peptide K21 (Leu 365-Lys 385), thus corrobo- 5.5.3. Location of Tyr-378 and Tyr-383 - Examination rating our previous conclusion that K21 is a part of the of the structure further reveals that Tyr-378 is located at a enzyme’s active center (Papers II and III). One patch of suitable distance from an LTA4 molecule modeled into the the cavity is hydrophilic, with Gln-134, Asp-375, and the hydrophobic cavity to allow participation in covalent hydroxyl group of Tyr-267 clustering together. If LTA4 is binding/inactivation of the enzyme. In fact, in this model modeled into this pocket such that the 5,6-epoxide moiety for substrate binding, the position of Tyr 378 in the LTA4 is bound to Zn2+, then C-7 to C-20 of the fatty acid back- binding pocket gets near the C-6 atom of LTA4. One may bone of LTA4 fits snugly into the deeper cavity, adopting thus speculate that the phenolic hydroxyl can attack the a bent conformation (Fig. 11). Furthermore, the C-1 car- carbocation intermediate to possibly form an ether adduct boxylate could make direct electrostatic interactions with at C-6, in agreement with the notion that LTA4 acts as an the positive charges of the conserved Arg-563 and Lys- affinity label (Fig. 12). However, Tyr-383 is also near C-6 565, in agreement with the fact that the free carboxylic of the carbocation intermediate and could potentially react acid of LTA4 is required for catalysis. Based on these in a similar manner. In fact, suicide inactivation of LTA4 findings and previous biochemical data, a mechanism for hydrolase with the alternative substrate LTA , a double the epoxide hydrolase reaction can be proposed (Fig. 11). 3 bond isomer of LTA , was recently shown to occur via Thus, assuming that LTA binds to the hydrophobic cav- 4 4 formation of an ether adduct at Tyr-383 [107]. In this ity, as described above, the catalytic zinc gets proximal to study, it was shown that the conjugated triene was pre- the labile allylic epoxide, suggesting that Zn2+ acts as a served in the LTA –protein adduct, which in turn demon- weak Lewis acid to activate and open the epoxide ring 3 strates that the ether bond had to be formed either at C-6 (Fig. 11). A carbocation would be generated whose charge or C-12. Considering the proposed model for binding of is delocalized over the conjugated triene system (C-6 to LTA (see above), these data in fact support a model that C-12), leaving the planar sp2 hybridized C-12 open for 4 involves coupling of a tyrosine at C-6 of the carbocation nucleophilic attack from either side of the molecule. In this model, any of the residues Gln-134, Tyr-267 or Asp- intermediate (Fig. 12). Hence, it appears likely that any of 375 is positioned to polarize a water molecule for attack the two tyrosines (Tyr-378 and Tyr-383) may become at C-12 and could thus control the positional and stereo- covalently modified depending on the structure of the leukotriene epoxide and its alignment in the substrate specific insertion of the 12R-hydroxyl group in LTB4. binding pocket. Moreover, the shape and curvature of the LTA4 bind- ing cavity also suggest the chemical strategy for creation The structure also provides some information regarding the function of Tyr-378 and Tyr-383. Thus, both residues of the 6-cis double bond in LTB4. Since there is free rota- are hydrogen-bonded to each other and in our model they tion between C-6 and C-7 of LTA4, the enzyme may keep this bond in a “pro-cis” configuration in the transition get in close contact with the conjugated triene system just state, which would promote the formation of a cis double at the angle of the L-shaped binding cavity. In fact, their bond from the carbocation intermediate (Fig. 11). The positions would become ideal for assisting optimal

25 Martin J. Mueller

295 Tyr378 His His299

Glu318 2+ Zn O O O C6 -+BH O

A- C12

295 Tyr378 His 299 His 318 Glu 2+ Zn + O - H O O

H H C6 +BH - O + O A C12

295 His His299 Gul 318 378 2+ Tyr Zn

H O H O OH O C6 -+BH H H O - O A C12

Fig. 12. Proposed reaction mechanism for covalent modification of Tyr-378 during suicide inactivation of LTA4 hydrolase. substrate alignment and promoting a specific double bond differential peptide mapping (Paper II), mutational analy- configuration, in line with what has been proposed from sis and characterizations of catalytic variants of the en- mutational analysis and catalytic properties of [Y378F] zyme generated by protein engineering (Papers III-V). and [Y383Q]LTA4 hydrolases (Papers III, IV, [10]). Further details regarding the molecular events involved in substrate-mediated inactivation of LTA4 hydrolase and its 5.5.4. Concluding remarks - Overall, it is very satisfy- relation to substrate binding and catalysis will hopefully ing to see how well our biochemical data agree with the be obtained from future structures of inhibited enzyme structural information obtained by X-ray crystallography, species. including the results of chemical modification (Paper I),

26

References

6. ACKNOWLEDGEMENTS 7. REFERENCES

I wish to express my sincere gratitude to: 1. Abramovitz, M., Wong, E., Cox, M.E., Richardson, C.D., Professor Bengt Samuelsson who has provided excellent Li, C. and Vickers, P.J. (1993) 5-lipoxygenase-activating working facilities and a stimulating work environment. protein stimulates the utilization of arachidonic acid by 5- lipoxygenase. Eur. J. Biochem., 215, 105-111. My tutor Jesper Haeggström for introducing me into the 2. Aharony, D., Redkar-Brown, D.G., Hubbs, S.J. and Stein, R.L. (1987) Kinetic studies on the inactivation of 5- world of eicosanoids, his continuous enthusiasm, support, lipoxygenase by 5(S)-hydroperoxyeicosatetraenoic acid. stimulating discussions, and criticism. It was really pleas- Prostaglandins, 33, 85-100. ant to share the moments of success and disappointment, 3. Ali, A., Ford-Hutchinson, A.W. and Nicholson, D.W. and I look forward to a continued collaboration with him. (1994) Activation of protein kinase C down-regulates leu- I also enjoyed the late night discussions about science, life kotriene C4 synthase activity and attenuates cysteinyl leu- and everything. Jesper finally persuaded me to defend my kotriene production in an eosinophilic substrain of HL-60 work as a thesis. Without his encouragement this thesis cells. J. Immunol., 153, 776-88. would have been never completed. 4. Andberg, M., Wetterholm, A., Medina, J.F. and Haegg- strom, J.Z. (2000) Leukotriene A4 hydrolase: a critical I am especially grateful to Martina Andberg, who intro- role of -296 for the binding of bestatin. Bio- duced me into the practice of molecular biology. Martina chem. J., 345, 621-5. taught me the virtue of patience (at least temporarily with 5. Bailie, M.B., Standiford, T.J., Laichalk, L.L., Coffey, M.J., Strieter, R. and Peters-Golden, M. (1996) Leukot- success). Apart from being a nice and pleasant lab com- riene-deficient mice manifest enhanced lethality from panion, she eventually became a friend of my family. Our Klebsiella pneumonia in association with decreased alveo- holidays at her family’s island in Finland will never be lar phagocytic and bactericidal activities. J. forgotten. Immunol., 157, 5221-5224. 6. Barret, A.J., Rawlings, N.D. and Woessner, J.F. (1998) Siobhan O’Sullivan for friendship, refreshing - not neces- Family M1 of membrane alanyl aminopeptidase. In Hand- sarily scientific - conversations and for drawing my atten- book of proteolytic enzymes, (Barret, A.J., Rawlings, N.D. tion to isoprostanoids, which eventually became a center and Woessner, J.F., eds.), pp. 994-996. Academic Press, of my scientific endeavors in Munich and Wuerzburg. London, San Diego. 7. Baset, H.A., Ford-Hutchinson, A.W. and O'Neill, G.P. Eva Ohlson for sharing her knowledge and her patience (1998) Molecular cloning and functional expression of a Caenorhabditis elegans aminopeptidase structurally re- with a somewhat inpatient chemist. lated to mammalian leukotriene A4 hydrolases. J. Biol. Chem., 273, 27978-27987. Anders Wetterholm, for generous help whenever it was 8. Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, needed and for nice company inside and outside of KI. G.J. (2001) Role for a bidentate ribonuclease in the initia- tion step of RNA interference. Nature, 409, 363-366. My German colleague Udo Oppermann for his valuable 9. Bigby, T.D., Levy, B.D. and Serhan, C.N. (1998) Cell contributions to this work and his pleasant company in biology of the 5-lipoxygenase pathway. Amplification and Sweden. generation of leukotrienes and lipoxins by transcellular biosynthesis. In 5-lipoxygenase products in asthma, (Hol- A special thank is due to Karin Demin and Ylva Blom- gate, S. and Dahlén, S.-E., eds.), pp. 125-173. Marcel berg, superb secretaries at Kemi II, for their logistic help Dekker, inc., New York. and all their efforts to make my stay in Stockholm com- 10. Blomster Andberg, M., Hamberg, M. and Haeggström, J.Z. (1997) Mutation of tyrosine 383 in leukotriene A fortable and pleasant. 4 hydrolase allows formation of 5S,6S-dihydroxy-7,9-trans- 11,14-cis-eicosatetraenoic acid: Implications for the epox- A big thank you goes to all the colleagues who provided a ide hydrolase mechanism. J. Biol. Chem., 272, 23057- friendly lab atmosphere, help, advice, and company at 23063. work: 11. Blomster, M., Wetterholm, A., Mueller, M.J. and Haegg- Per-Johan Jakobsson, Filippa Kull, Maria Kumlin, Agneta strom, J.Z. (1995) Evidence for a catalytic role of tyrosine Nordberg, Pontus Larsson, Stina Feltenmark, and Tove 383 in the peptidase reaction of leukotriene A4 hydrolase. Hammarberg Eur. J. Biochem., 231, 528-534. 12. Bonventre, J.V., Huang, Z., Taheri, M.R., O'Leary, E., Li, Finally, I wish to express my gratitude to my family and E., Moskowitz, M.A. and Sapirstein, A. (1997) Reduced many friends for their support fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A . Nature, 390, 622-625. 2 13. Borgeat, P. and Samuelsson, B. (1979) Arachidonic acid metabolism in polymorphonuclear leukocytes: Unstable intermediate in formation of dihydroxy acids. Proc. Natl. Acad. Sci. USA, 76, 3213-3217.

27 Martin J. Mueller

14. Borgeat, P. and Samuelsson, B. (1979) Transformation of 29. Dennis, E.A. (1997) The growing phospholipase A2 su- arachidonic acid by rabbit polymorphonuclear leukocytes. perfamily of signal transduction enzymes. TiBS, 22, 1-2. Formation of a novel dihydroxyeicosatetraenoic acid. J. 30. Dessen, A., Tang, J., Schmidt, H., Stahl, M., Clark, J.D., Biol. Chem., 254, 2643-2646. Seehra, J. and Somers, W.S. (1999) Crystal structure of 15. Boyington, J.C., Gaffney, B.J. and Amzel, L.M. (1993) human cytosolic phospholipase A2 reveals a novel topol- The three-dimensional structure of an arachidonic acid 15- ogy and catalytic mechanism. Cell, 97, 349-60. lipoxygenase. Science, 260, 1482-1486. 31. Devchand, P.R., Keller, H., Peters, J.M., Vazquez, M., 16. Bradford, M.M. (1976) A rapid and sensitive method for Gonzalez, F.J. and Wahli, W. (1996) The PPARα- the quantitation of microgram quantities of protein utiliz- leukotriene B4 pathway to inflammation control. Nature, ing the principle of protein-dye binding. Anal. Biochem., 384, 39-43. 72, 248-254. 32. DeWitt, D.L. and Smith, W.L. (1983) Purification of 17. Brock, T.G., Anderson, J.A., Fries, F.P., Peters-Golden, prostacyclin synthase from bovine aorta by immunoaffin- M. and Sporn, P.H. (1999) Decreased leukotriene C4 syn- ity chromatography. Evidence that the enzyme is a hemo- thesis accompanies adherence-dependent nuclear import protein. J. Biol. Chem., 258, 3285-93. of 5-lipoxygenase in human blood eosinophils. J. Immu- 33. Dixon, R.A.F., Diehl, R.E., Opas, E., Rands, E., Vickers, nol., 162, 1669-76. P.J., Evans, J.F., Gillard, J.W. and Miller, D.K. (1990) 18. Brock, T.G., McNish, R.W., Bailie, M.B. and Peters- Requirement of a 5-lipoxygenase-activating protein for Golden, M. (1997) Rapid import of cytosolic 5- leukotriene synthesis. Nature, 343, 282-284. lipoxygenase into the nucleus of neutrophils after in vivo 34. Dixon, R.A.F., Jones, R.E., Diehl, R.E., Bennett, C.D., recruitment and in vitro adherence. J. Biol. Chem., 272, Kargman, S. and Rouzer, C.A. (1988) Cloning of the 8276-8280. cDNA for human 5-lipoxygenase. Proc. Natl. Acad. Sci. 19. Brock, T.G., McNish, R.W. and Peters-Golden, M. (1995) USA, 85, 416-420. Translocation and leukotriene synthetic capacity of nu- 35. Drazen, J.F., Israel, E. and O'Byrne, P. (1999) Treatment clear 5-lipoxygenase in rat basophilic leukemia cells and of asthma with drugs modifying the leukotriene pathway. alveolar macrophages. J. Biol. Chem., 270, 21652-21658. N. Engl. J. Med., 340, 197-206. 20. Byrum, R.S., Goulet, J.L., Griffiths, R.J. and Koller, B.H. 36. Drazen, J.M., Yandava, C.N., Dube, L., Szczerback, N., (1997) Role of the 5-lipoxygenase-activating protein Hippensteel, R., Pillari, A., Israel, E., Schork, N., Silver- (FLAP) in murine acute inflammatory responses. J. Exp. man, E.S., Katz, D.A. et al. (1999) Pharmacogenetic asso- Med., 185, 1065-1075. ciation between ALOX5 promoter genotype and the re- 21. Byrum, R.S., Goulet, J.L., Snouwaert, J.N., Griffiths, R.J. sponse to anti-asthma treatment. Nature Gen., 22, 168-70. and Koller, B.H. (1999) Determination of the contribution 37. Evans, J.F. and Kargman, S. (1992) Bestatin inhibits 3 of cysteinyl leukotrienes and leukotriene B4 in acute in- covalent coupling of [ H]LTA4 to human leukocyte LTA4 flammatory responses using 5-lipoxygenase- and leukot- hydrolase. FEBS Lett., 297, 139-142. riene A4 hydrolase-deficient mice. J. Immunol., 163, 38. Evans, J.F., Nathaniel, D.J., Zamboni, R.J. and Ford- 6810-6819. Hutchinson, A.W. (1985) Leukotriene A3: A poor sub- 22. Chen, X.S. and Funk, C.D. (2001) The N-terminal "beta- strate but a potent inhibitor of rat and human neutrophil barrel" domain of 5-lipoxygenase is essential for nuclear leukotriene A4 hydrolase. J. Biol. Chem., 260, 10966- membrane translocation. J. Biol. Chem., 276, 811-8. 10970. 23. Chen, X.S., Sheller, J.R., Johnson, E.N. and Funk, C.D. 39. Fersht, A. (1984) Active-site-directed and enzyme- (1994) Role of leukotrienes revealed by targeted disrup- activated irreversible inhibitors: "Affinity labels" and "sui- tion of the 5-lipoxygenase gene. Nature, 372, 179-82. cide inhibitors". In Enzyme structure and mechanism., pp. 24. Clark, J.D., Lin, L.-L., Kriz, R.W., Ramesha, C.S., Sultz- 248-260. W.H. Freeman and Company, New York. man, L.A., Lin, A.Y., Milona, N. and Knopf, J.L. (1991) 40. Fitzpatrick, F.A., Haeggström, J., Granström, E. and Sa- A novel arachidonic acid-selective cytosolic PLA2 con- muelsson, B. (1983) Metabolism of leukotriene A4 by an tains a Ca2+-dependent translocation domain with homol- enzyme in blood plasma: A possible leukotactic mecha- ogy to PKC and GAP. Cell, 65, 1043-1051. nism. Proc. Natl. Acad. Sci. USA, 80, 5425-5429. 25. Clark, J.D., Milona, N. and Knopf, J.L. (1990) Purifica- 41. Fitzpatrick, F.A., Morton, D.R. and Wynalda, M.A. tion of a 110-kilodalton cytosolic phospholipase A2 from (1982) Albumin stabilizes leukotriene A4. J. Biol. Chem., the human monocytic cell line U937. Proc. Natl. Acad. 257, 4680-4683. Sci. USA, 87, 7708-7712. 42. Ford-Hutchinson, A.W., Gresser, M. and Young, R.N. 26. Cowburn, A.S., Sladek, K., Soja, J., Adamek, L., Ni- (1994) 5-Lipoxygenase. [Review]. Ann. Rev. Biochem., zankowska, E., Szczeklik, A., Lam, B.K., Penrose, J.F., 63, 383-417. Austen, F.K., Holgate, S.T. et al. (1998) Overexpression 43. Funk, C.D. (1996) The molecular biology of mammalian of leukotriene C4 synthase in bronchial biopsies from pa- lipoxygenases and the quest for eicosanoid functions using tients with aspirin-intolerant asthma. J. Clin. Invest., 101, lipoxygenase-deficient mice. [Review] [139 refs]. Bio- 834-46. chim. Biophys. Acta, 1304, 65-84. 27. De Carolis, E., Denis, D. and Riendeau, D. (1996) Oxida- 44. Funk, C.D., Hoshiko, S., Matsumoto, T., Rådmark, O. and tive inactivation of human 5-lipoxygenase in phosphati- Samuelsson, B. (1989) Characterization of the human 5- dylcholine vesicles. Eur. J. Biochem., 235, 416-23. lipoxygenase gene. Proc. Natl. Acad. Sci. USA, 86, 2587- 28. de Carvalho, M.G., McCormack, A.L., Olson, E., Gho- 2591. mashchi, F., Gelb, M.H., Yates, J.R., 3rd and Leslie, C.C. 45. Gan, Q.F., Witkop, G.L., Sloane, D.L., Straub, K.M. and (1996) Identification of phosphorylation sites of human Sigal, E. (1995) Identification of a specific methionine in 85-kDa cytosolic phospholipase A2 expressed in insect mammalian 15-lipoxygenase which is oxygenated by the cells and present in human monocytes. J. Biol. Chem., enzyme product 13-HPODE: dissociation of sulfoxide 271, 6987-97.

28

References

formation from self-inactivation. Biochemistry, 34, 7069- calcium and mediates calcium stimulation of enzyme ac- 79. tivity. J. Biol. Chem., 275, 38787-93. 46. Gijon, M.A., Spencer, D.M., Kaiser, A.L. and Leslie, C.C. 61. Hammarberg, T., Zhang, Y.Y., Lind, B., Rådmark, O. and (1999) Role of phosphorylation sites and the C2 domain Samuelsson, B. (1995) Mutations at the C-terminal isoleu- in regulation of cytosolic phospholipase A2. J. Cell Biol., cine and other potential iron ligands of 5-lipoxygenase. 145, 1219-32. Eur. J. Biochem., 230, 401-7. 47. Gillmor, S.A., Villaseñor, A., Fletterick, R., Sigal, E. and 62. Haribabu, B., Verghese, M.W., Steeber, D A, Sellars, Browner, M. (1997) The structure of mammalian 15- D.D., Bock, C.B. and Snyderman, R. (2000) Targeted dis- lipoxygenase reveals similarity to the lipases and the de- ruption of the leukotriene B4 receptor in mice reveals its terminants of substrate specificity. Nature Struct. Biol., 4, role in inflammation and platelet-activating factor-induced 1003-1009. anaphylaxis. J. Exp. Med., 192, 433-438. 48. Glover, S., de Carvalho, M.S., Bayburt, T., Jonas, M., Chi, 63. Hartel, B., Ludwig, P., Schewe, T. and Rapoport, S.M. E., Leslie, C.C. and Gelb, M.H. (1995) Translocation of (1982) Self-inactivation by 13-hydroperoxylinoleic acid the 85-kDa phospholipase A2 from cytosol to the nuclear and lipohydroperoxidase activity of the reticulocyte li- envelope in rat basophilic leukemia cells stimulated with poxygenase. Eur. J. Biochem., 126, 353-7. calcium ionophore or Ige/antigen. J. Biol. Chem., 270, 64. Healy, A.M., Peters-Golden, M., Yao, J.P. and Brock, 15359-15367. T.G. (1999) Identification of a bipartite nuclear localiza- 49. Goulet, J.L., Byrum, R.S., Key, M.L., Nguyen, M., Wag- tion sequence necessary for nuclear import of 5- oner, V.A. and Koller, B.H. (2000) Genetic factors deter- lipoxygenase. J. Biol. Chem., 274, 29812-8. mine the contribution of leukotrienes to acute inflamma- 65. Hecker, M. and Ullrich, V. (1989) On the mechanism of tory responses. J. Immunol., 164, 4899-907. prostacyclin and thromboxane A2 biosynthesis. J. Biol. 50. Goulet, J.L., Griffiths, R.C., Ruiz, P., Spurney, R.F., Chem., 264, 141-50. Pisetsky, D.S., Koller, B.H. and Coffman, T.M. (1999) 66. Heise, C.E., O'Dowd, B.F., Figueroa, D.J., Sawyer, N., Deficiency of 5-lipoxygenase abolishes sex-related sur- Nguyen, T., Im, D.-S., Stocco, R., Bellefeuille, J.N., vival differences in MRL-lpr/lpr mice. J. Immunol., 163, Abramovitz, M., Cheng Jr. Williams, R. et al. (2000) 359-66. Characterization of the human cysteinyl leukotriene 2 51. Goulet, J.L., Snouwaert, J.N., Latour, A.M., Coffman, (CysLT2) receptor. J. Biol. Chem., 275, 30531-30536. T.M. and Koller, B.H. (1994) Altered inflammatory re- 67. Hill, E., Maclouf, J., Murphy, R.C. and Henson, P.M. sponses in leukotriene-deficient mice. Proc. Natl. Acad. (1992) Reversible membrane association of neutrophil 5- Sci. USA, 91, 12852-6. lipoxygenase is accompanied by retention of activity and a 52. Griffin, K.J., Gierse, J., Krivi, G. and Fitzpatrick, F.A. change in substrate specificity. J. Biol. Chem., 267, (1992) Opioid peptides are substrates for the bifunctional 22048-22053. enzyme LTA4 hydrolase/aminopeptidase. Prostaglandins, 68. Hogg, J.H., Ollmann, I.R., Haeggström, J.Z., Wetterholm, 44, 251-257. A., Samuelsson, B. and Wong, C.-H. (1995) Amino hy- 53. Griffiths, R.J., Smith, M.A., Roach, M.L., Stock, J.L., droxamic acids as potent inhibitors of LTA4 hydrolase. Stam, E.J., Milici, A.J., Scampoli, D.N., Eskra, J.D., By- Bioorg. Med. Chem., 3, 1405-1415. rum, R.S., Koller, B.H. et al. (1997) Collagen-induced 69. Hsieh, F.H., Lam, B.K., Penrose, J.F., Austen, K.F. and arthritis is reduced in 5-lipoxygenase-activating protein- Boyce, J.A. (2001) T helper cell type 2 cytokines coordi- deficient mice. J. Exp. Med., 185, 1123-1129. nately regulate immunoglobulin E-dependent cysteinyl 54. Gupta, N., Nicholson, D.W. and Ford-Hutchinson, A.W. leukotriene production by human cord blood-derived mast (1999) Demonstration of cell-specific phosphorylation of cells: profound induction of leukotriene C4 synthase ex- LTC4 synthase. FEBS Lett., 449, 66-70. pression by interleukin 4. J. Exp. Med., 193, 123-33. 55. Gut, J., Goldman, D.W., Jamieson, G.C. and Trudell, J.R. 70. In, K.H., Asano, K., Beier, D., Grobholz, J., Finn, P.W., (1987) Conversion of leukotriene A4 to leukotriene B4: Silverman, E.K., Silverman, E.S., Collins, T., Fischer, Catalysis by human liver microsomes under anaerobic A.R., Keith, T.P. et al. (1997) Naturally occurring muta- conditions. Arch. Biochem. Biophys., 259, 497-509. tions in the human 5-lipoxygenase gene promoter that 56. Haeggström, J.Z. (1997) The molecular biology of the modify transcription factor binding and reporter gene tran- leukotriene A4 hydrolase. In SRS-A to leukotrienes, (Ste- scription. J. Clin. Invest., 99, 1130-7. ven Holgate and Dahlén, S.-E., eds.), pp. 85-100. Black- 71. Irvin, C.G., Tu, Y.P., Sheller, J.R. and Funk, C.D. (1997) well Science Ltd, Oxford. 5-lipoxygenase products are necessary for ovalbumin- 57. Haeggström, J.Z., Wetterholm, A., Shapiro, R., Vallee, induced airway responsiveness in mice. Am. J. Physiol., B.L. and Samuelsson, B. (1990) Leukotriene A4 hy- 16, L1053-L1058. drolase: A zinc metalloenzyme. Biochem. Biophys. Res. 72. Ishii, S., Noguchi, M., Miyano, M., Matsumoto, T. and Commun., 172, 965-970. Noma, M. (1992) Mutagenesis studies on the amino acid 58. Haeggström, J.Z., Wetterholm, A., Vallee, B.L. and residues involved in the iron-binding and the activity of Samuelsson, B. (1990) Leukotriene A4 hydrolase: An human 5-lipoxygenase. Biochem. Biophys. Res. Commun., epoxide hydrolase with peptidase activity. Biochem. Bio- 182, 1482-1490. phys. Res. Commun., 173, 431-437. 73. Ishizaka, N., Nakao, A., Ohishi, N., Suzuki, M., Aizawa, 59. Hall, E.R., Tuan, W.M. and Venton, D.L. (1986) Produc- T., Taguchi, J., Nagai, R., Shimizu, T. and Ohno, M. tion of platelet thromboxane A2 inactivates purified hu- (1999) Increased leukotriene A4 hydrolase expression in man platelet thromboxane synthase. Biochem. J., 233, the heart of angiotensin II-induced hypertensive rat. FEBS 637-41. Lett., 463, 155-9. 60. Hammarberg, T., Provost, P., Persson, B. and Radmark, 74. Jakobsson, P.J., Mancini, J.A. and Ford-Hutchinson, A.W. O. (2000) The N-terminal domain of 5-lipoxygenase binds (1996) Identification and characterization of a novel hu- man microsomal glutathione S-transferase with leukot-

29 Martin J. Mueller

riene C4 synthase activity and significant sequence iden- (1992) ω-[(ω-arylalkyl)aryl]alkanoic acids: A new class of tity to 5-lipoxygenase-activating protein and leukotriene specific LTA hydrolase inhibitors. J. Med. Chem., 35, 4 C4 synthase. J. Biol. Chem., 271, 22203-22210. 3156-3169. 75. Jakobsson, P.J., Mancini, J.A., Riendeau, D. and Ford- 91. Laemmli, U.K. (1970) Cleavage of structural proteins Hutchinson, A.W. (1997) Identification and characteriza- during the assembly of the head of bacteriophage T4. Na- tion of a novel microsomal enzyme with glutathione- ture, 227, 680-685. dependent transferase and peroxidase activities. J. Biol. 92. Lam, B.K., Penrose, J.F., Freeman, G.J. and Austen, K.F. Chem., 272, 22934-9. (1994) Expression cloning of a cDNA for human leukot- 76. Jones, D.A. and Fitzpatrick, F.A. (1990) "Suicide" inacti- riene C4 synthase, an integral membrane protein conjugat- vation of thromboxane A2 synthase. Characteristics of ing reduced glutathione to leukotriene A4. Proc. Natl. mechanism-based inactivation with isolated enzyme and Acad. Sci. USA, 91, 7663-7. intact platelets. J. Biol. Chem., 265, 20166-71. 93. Lam, B.K., Penrose, J.F., Xu, K.Y., Baldasaro, M.H. and 77. Jones, D.A. and Fitzpatrick, F.A. (1991) Thromboxane A2 Austen, K.F. (1997) Site-directed mutagenesis of human synthase. Modification during "suicide" inactivation. J. leukotriene C4 synthase. J. Biol. Chem., 272, 13923- Biol. Chem., 266, 23510-4. 13928. 78. Kamohara, M., Takasaki, J., Matsumoto, M., Saito, T., 94. Lecomte, M., Lecocq, R., Dumont, J.E. and Boeynaems, Ohishi, T., Ishii, H. and Furuichi, K. (2000) Molecular J.M. (1990) Covalent binding of arachidonic acid metabo- cloning and characterization of another leukotriene B4 re- lites to human platelet proteins. Identification of pros- ceptor. J. Biol. Chem., 275, 27000-27004. taglandin H synthase as one of the modified substrates. J. 79. Kanaoka, Y., Maekawa, A., Penrose, J.F., Austen, K.F. Biol. Chem., 265, 5178-87. and Lam, B.K. (2001) Attenuated zymosan-induced peri- 95. Lepley, R.A. and Fitzpatrick, F.A. (1994) 5-Lipoxygenase toneal vascular permeability and IgE dependent passive contains a functional Src homology 3-binding motif that cutaneous anaphylaxis in mice lacking leukotriene C4 syn- interacts with the Src homology 3 domain of Grb2 and cy- thase. J. Biol. Chem., 276, 22608-22613. toskeletal proteins. J. Biol. Chem., 269, 24163-8. 80. Kargman, S., Vickers, P.J. and Evans, J.F. (1992) 96. Lepley, R.A. and Fitzpatrick, F.A. (1994) Irreversible A23187-induced translocation of 5-lipoxygenase in os- inactivation of 5-lipoxygenase by leukotriene A4. Charac- teosarcoma cells. J. Cell Biol., 119, 1701-9. terization of product inactivation with purified enzyme 81. Kato, K., Yokomizu, T., Izumi, T. and Shimizu, T. (2000) and intact leukocytes. J. Biol. Chem., 269, 2627-31. Cell-specific transcriptional regulation of human leukot- 97. Lepley, R.A. and Fitzpatrick, F.A. (1998) 5-Lipoxygenase riene B4 receptor gene. J. Exp. Med., 192, 413-420. compartmentalization in granulocytic cells is modulated 82. Kennedy, B.P., Diehl, R.E., Boie, Y., Adam, M. and by an internal bipartite nuclear localizing sequence and Dixon, R.A.F. (1991) Gene characterization and promoter nuclear factor kappa B complex formation. Arch. Bio- analysis of the human 5-lipoxygenase-activating protein chem. Biophys., 356, 71-6. (FLAP). J. Biol. Chem., 266, 8511-8516. 98. Lepley, R.A., Muskardin, D.T. and Fitzpatrick, F.A. 83. Kester, W.R. and Matthews, B.W. (1977) Crystallographic (1996) Tyrosine kinase activity modulates catalysis and study of the binding of dipeptide inhibitors to thermolysin: translocation of cellular 5-lipoxygenase. J. Biol. Chem., Implications for the mechanism of catalysis. Biochemistry, 271, 6179-6184. 16, 2506-2516. 99. Lewis, R.A., Austen, K.F. and Soberman, R.J. (1990) 84. Kishimoto, K., Nakamura, M., Suzuki, H., Yoshimoto, T., Leukotrienes and other products of the 5-lipoxygenase Yamamoto, S., Takao, T., Shimonishi, Y. and Tanabe, T. pathway. New Engl. J. Med., 323, 645-655. (1996) Suicide inactivation of porcine leukocyte 12- 100. Lin, L.-L., Wartman, M., Lin, A.Y., Knopf, J.L., Seth, A. lipoxygenase associated with its incorporation of 15- and Davis, R.J. (1993) cPLA2 is phosphorylated and acti- hydroperoxy-5,8,11,13-eicosatetraenoic acid derivative. vated by MAP kinase. Cell, 72, 269-278. Biochim. Biophys. Acta, 1300, 56-62. 101. Lundberg, K.S., Shoemaker, D.D., Adams, M.W., Short, 85. Kramer, R.M., Roberts, E.F., Manetta, J. and Putnam, J.E. J.M., Sorge, J.A. and Mathur, E.J. (1991) High-fidelity 2+ (1991) The Ca - sensitive cytosolic phospholipase A2 is amplification using a thermostable DNA polymerase iso- a 100-kDa protein in human monoblast U937 cells. J. lated from Pyrococcus furiosus. Gene, 108, 1-6. Biol. Chem., 266, 5268-5272. 102. Lynch, K.R., O'Neill, G.P., Liu, Q., Im, D.S., Sawyer, N., 86. Kull, F., Ohlson, E. and Haeggstrom, J.Z. (1999) Cloning Metters, K.M., Coulombe, N., Abramovitz, M., Figueroa, and characterization of a bifunctional leukotriene A4 hy- D.J., Zeng, Z. et al. (1999) Characterization of the human drolase from Saccharomyces cerevisiae. J. Biol. Chem., cysteinyl leukotriene CysLT1 receptor. Nature, 399, 789- 274, 34683-34690. 93. 87. Kull, F., Ohlson, E., Lind, B. and Haeggström, J.Z. (2001) 103. Malaviya, R. and Jakschik, B.A. (1993) Reversible trans- Saccharomyces cerevisiae leukotriene A4 hydrolase, for- location of 5-lipoxygenase in mast cells upon IgE/antigen mation of leukotriene B4 and identification of catalytic stimulation. J. Biol. Chem., 268, 4939-44. residues. Biochemistry, , in press. 104. Malfroy, B., Kado-Fong, H., Gros, C., Giros, B., 88. Kulmacz, R.J. (1987) Attachment of substrate metabolite Schwartz, J.-C. and Hellmiss, R. (1989) Molecular cloning to prostaglandin H synthase upon reaction with arachi- and amino acid sequence of rat kidney aminopeptidase M: donic acid. Biochem. Biophys. Res. Commun., 148, 539- A member of a super family of zinc-metallohydrolases. 45. Biochem. Biophys. Res. Commun., 161, 236-241. 89. Kyte, J. (1995) Modification and labeling of the active 105. Mancini, J.A., Abramovitz, M., Cox, M.E., Wong, E., site. In Mechanism in protein chemistry., pp. 314-344. Charleson, S., Perrier, H., Wang, Z., Prasit, P. and Vick- Garland Publishing Inc., New York, London. ers, P.J. (1993) 5-lipoxygenase-activating protein is an 90. Labaudinière, R., Hilboll, G., Leon-Lomeli, A., Lauten- arachidonate binding protein. FEBS Lett., 318, 277-281. schläger, H.-H., Parnham, M., Kuhl, P. and Dereu, N.

30

References

106. Mancini, J.A. and Evans, J.F. (1995) Cloning and charac- active site iron and its ligands in soybean lipoxygenase L- terization of the human leukotriene A4 hydrolase gene. 1. Biochemistry, 32, 6320-6323. Eur. J. Biochem., 231, 65-71. 121. Miyashita, A., Crystal, R.G. and Hay, J.G. (1995) Identifi- 107. Mancini, J.A., Waugh, R.J., Thompson, J.A., Evans, J.F., cation of a 27 bp 5'-flanking region element responsible Belley, M., Zamboni, R. and Murphy, R.C. (1998) Struc- for the low level constitutive expression of the human cy- tural characterization of the covalent attachment of leukot- tosolic phospholipase A2 gene. Nucleic Acids Res., 23, riene A3 to leukotriene A4 hydrolase. Arch. Biochem. Bi- 293-301. ophys., 354, 117-24. 122. Montero, A., Nassar, G.M., Uda, S., Munger, K.A. and 108. Mancuso, P., Nana-Sinkam, P. and Peters-Golden, M. Badr, K.F. (2000) Reciprocal regulation of LTA4 hy- (2001) Leukotriene B4 augments neutrophil phagocytosis drolase expression in human monocytes by gamma- of Klebsiella pneumoniae. Infection & Immunity, 69, interferon and interleukins 4 and 13: potential relevance to 2011-6. leukotriene regulation in glomerular disease. Exp. 109. Marshall, L.A., Bolognese, B., Winkler, J.D. and Roshak, Nephrol., 8, 258-65. A. (1997) Depletion of human monocyte 85-kDa phos- 123. Munafo, D.A., Shindo, K., Baker, J.R. and Bigby, T.D. pholipase A2 does not alter leukotriene formation. J. Biol. (1994) Leukotriene A4 hydrolase in human bronchoalveo- Chem., 272, 759-65. lar lavage fluid. J. Clin. Invest., 93, 1024-1050. 110. Marshall, P.J., Kulmacz, R.J. and Lands, W.E. (1987) 124. Murakami, M., Austen, K.F., Bingham, C.O., Friend, Constraints on prostaglandin biosynthesis in tissues. J. D.S., Penrose, J.F. and Arm, J.P. (1995) Interleukin-3 Biol. Chem., 262, 3510-7. regulates development of the 5-lipoxygenase/leukotriene 111. Matsumoto, T., Funk, C.D., Rådmark, O., Höög, J.-O., C4 synthase pathway in mouse mast cells. J. Biol. Chem., Jörnvall, H. and Samuelsson, B. (1988) Molecular cloning 270, 22653-22656. and amino acid sequence of human 5-lipoxygenase. Proc. 125. Murakami, M., Penrose, J.F., Urade, Y., Austen, K.F. and Natl. Acad. Sci. USA, 85, 26-30; published erratum in Arm, J.P. (1995) Interleukin 4 suppresses c-kit ligand- Proc. Natl. Acad. Sci. USA 85: 3406. induced expression of cytosolic phospholipase A2 and 112. Mayatepek, E. and Flock, B. (1998) Leukotriene C4- prostaglandin endoperoxide synthase 2 and their roles in synthesis deficiency: a new inborn error of metabolism separate pathways of eicosanoid synthesis in mouse bone linked to a fatal developmental syndrome. Lancet, 352, marrow-derived mast cells. Proc. Natl. Acad. Sci. USA, 1514-7. 92, 6107-6111. 113. Mayatepek, E., Lindner, M., Zelezny, R., Lindner, W., 126. Murphy, R.C., Hammarström, S. and Samuelsson, B. Brandstetter, G. and Hoffmann, G.F. (1999) A severely af- (1979) Leukotriene C: A slow reacting substance from fected infant with absence of cysteinyl leukotrienes in murine mastocytoma cells. Proc. Natl. Acad. Sci. USA, 76, cerebrospinal fluid: further evidence that leukotriene C4- 4275-4279. synthesis deficiency is a new neurometabolic disorder. 127. Nalefski, E.A., Sultzman, L.A., Martin, D.M., Kriz, R.W., Neuropediatrics, 30, 5-7. Towler, P.S., Knopf, J.L. and Clark, J.D. (1994) Delinea- 114. Maycock, A.L., Anderson, M.S., DeSousa, D.M. and tion of two functionally distinct domains of cytosolic 2+ Kuehl Jr, F.A. (1982) Leukotriene A4: Preparation and phospholipase A2, a regulatory Ca -dependent lipid- enzymatic conversion in a cell-free system to leukotriene binding domain and a Ca2+-independent catalytic domain. B4. J. Biol. Chem., 257, 13911-13914. J. Biol. Chem., 269, 18239-18249. 115. McGee, J. and Fitzpatrick, F. (1985) Enzymatic hydration 128. Narumiya, S., Sugimoto, Y. and Ushikubi, F. (1999) of leukotriene A4: Purification and characterization of a receptors: Structures, properties, and functions novel epoxide hydrolase from human erythrocytes. J. Biol. [Review]. Physiological Reviews, 79, 1193-1226. Chem., 260, 12832-12837. 129. Nguyen, T., Falgueyret, J.-P., Abramovitz, M. and Rien- 116. Medina, J.F., Wetterholm, A., Rådmark, O., Shapiro, R., deau, D. (1991) Evaluation of the role of conserved His Haeggström, J.Z., Vallee, B.L. and Samuelsson, B. (1991) and Met residues among lipoxygenases by site-directed Leukotriene A4 hydrolase: determination of the three zinc- mutagenesis of recombinant human 5-lipoxygenase. J. binding ligands by site directed mutagenesis and zinc Biol. Chem., 266, 22057-22062. analysis. Proc. Natl. Acad. Sci. USA, 88, 7620-7624. 130. Nicholson, D.W., Ali, A., Vaillancourt, J.P., Calaycay, 117. Miller, D.K., Gillard, J.W., Vickers, P.J., Sadowski, S., J.R., Mumford, R.A., Zamboni, R.J. and Ford-Hutchinson, Léveillé, C., Mancini, J.A., Charleson, P., Dixon, R.A.F., A.W. (1993) Purification to homogeneity and the N- Ford-Hutchinson, A.W., Fortin, R. et al. (1990) Identifica- terminal sequence of human leukotriene C4 synthase: A tion and isolation of a membrane protein necessary for homodimeric glutathione S-transferase composed of 18- leukotriene production. Nature, 343, 278-281. kDa subunits. Proc. Natl. Acad. Sci. USA, 90, 2015-2019. 118. Minami, M., Bito, H., Ohishi, N., Tsuge, H., Miyano, M., 131. Nicholson, D.W., Klemba, M.W., Rasper, D.M., Metters, Mori, M., Wada, H., Mutoh, H., Shimada, S., Izumi, T. et K.M., Zamboni, R.J. and Ford-Hutchinson, A.W. (1992) al. (1992) Leukotriene A4 hydrolase, a bifunctional en- Purification of human leukotriene C4 synthase from di- zyme. Distinction of leukotriene A4 hydrolase and amin- methylsulfoxide-differentiated U937 cells. Eur. J. Bio- opeptidase activities by site-directed mutagenesis at Glu- chem., 209, 725-734. 297. FEBS Lett., 309, 353-357. 132. Nilsson, N.E., Tryselius, Y. and Owman, C. (2000) Ge- 119. Minami, M., Ohishi, N., Mutoh, H., Izumi, T., Bito, H., nomic organization of the leukotriene B-4 receptor locus Wada, H., Seyama, Y., Toh, H. and Shimizu, T. (1990) of human chromosome 14. Biochem. Biophys. Res. Com- Leukotriene A4 hydrolase is a zinc-containing aminopep- mun., 274, 383-388. tidase. Biochem. Biophys. Res. Commun., 173, 620-626. 133. Nissen, J.B., Iversen, L. and Kragballe, K. (1995) Charac- 120. Minor, W., Steczko, J., Bolin, J.T., Otwinowski, Z. and terization of the aminopeptidase activity of epidermal leu- Axelrod, B. (1993) Crystallographic determination of the kotriene A4 hydrolase against the opioid dynorphin frag- ment 1-7. Br. J. Dermatol., 133, 742-749.

31 Martin J. Mueller

134. Noiri, E., Yokomizo, T., Nakao, A., Izumi, T., Fujita, T., polyclonal antibody. Am. J. Resp. Crit. Care Med., 152, Kimura, S. and Shimizu, T. (2000) An in vivo approach 283-289. showing the chemotactic activity of leukotriene B4 in 149. Percival, M.D. (1991) Human 5-lipoxygenase contains an acute renal ischemic-reperfusion injury. Proc. Natl. Acad. essential iron. J. Biol. Chem., 266, 10058-10061. Sci. USA, 97, 823-8. 150. Percival, M.D., Denis, D., Riendeau, D. and Gresser, M.J. 135. Nothacker, H.P., Wang, Z.W., Zhu, Y.H., Reinscheid, (1992) Investigation of the mechanism of non-turnover- R.K., Lin, S.H.S. and Civelli, O. (2000) Molecular clon- dependent inactivation of purified human 5-lipoxygenase. ing and characterization of a second human cysteinyl leu- Inactivation by H2O2 and inhibition by metal ions. Eur. J. kotriene receptor: Discovery of a subtype selective ago- Biochem., 210, 109-117. nist. Mol. Pharmacol., 58, 1601-1608. 151. Percival, M.D. and Ouellet, M. (1992) The characteriza- 136. Ohishi, N., Izumi, T., Minami, M., Kitamura, S., Seyama, tion of 5 histidine-serine mutants of human 5- Y., Ohkawa, S., Terao, S., Yotsumoto, H., Takaku, F. and lipoxygenase. Biochem. Biophys. Res. Commun., 186, Shimizu, T. (1987) Leukotriene A4 hydrolase in the hu- 1265-1270. man lung: Inactivation of the enzyme with leukotriene A4 152. Provost, P., Samuelsson, B. and Rådmark, O. (1999) isomers. J. Biol. Chem., 262, 10200-10205. Interaction of 5-lipoxygenase with cellular proteins. Proc. 137. Orning, L. and Fitzpatrick, F.A. (1992) Albumins activate Natl. Acad. Sci USA, 96, 1881-1885. peptide hydrolysis by the bifunctional enzyme LTA4 hy- 153. Puustinen, T., Scheffer, M.M. and Samuelsson, B. (1988) drolase/aminopeptidase. Biochemistry, 31, 4218-4223. Regulation of the human leukocyte 5-lipoxygenase: stimu- 138. Orning, L., Gierse, J., Duffin, K., Bild, G., Krivi, G. and lation by micromolar Ca2+ levels and phosphatidylcholine Fitzpatrick, F.A. (1992) Mechanism-based inactivation of vesicles. Biochim. Biophys. Acta, 960, 261-267. leukotriene A4 hydrolase/aminopeptidase by leukotriene 154. Rapoport, S., Hartel, B. and Hausdorf, G. (1984) Methion- A4. Mass spectrometric and kinetic characterization. J. ine sulfoxide formation: the cause of self-inactivation of Biol. Chem., 267, 22733-22739. reticulocyte lipoxygenase. Eur. J. Biochem., 139, 573-6. 139. Orning, L., Gierse, J.K. and Fitzpatrick, F.A. (1994) The 155. Richards, K.M. and Marnett, L.J. (1997) Leukocyte 12- bifunctional enzyme leukotriene A4 hydrolase is an argin- lipoxygenase: expression, purification, and investigation ine aminopeptidase of high efficiency and specificity. J. of the role of methionine residues in turnover-dependent Biol. Chem., 269, 11269-11273. inactivation and 5,8,11,14-eicosatetraynoic acid inhibi- 140. Orning, L., Jones, D.A. and Fitzpatrick, F.A. (1990) tion. Biochemistry, 36, 6692-9. Mechanism-based inactivation of leukotriene A4 hydrolase 156. Riddick, C.A., Serio, K.J., Hodulik, C.R., Ring, W.L., during leukotriene B4 formation by human erythrocytes. J. Regan, M.S. and Bigby, T.D. (1999) TGF-beta increases Biol. Chem., 265, 14911-14916. leukotriene C4 synthase expression in the monocyte-like 141. Orning, L., Krivi, G., Bild, G., Gierse, J., Aykent, S. and cell line, THP-1. J. Immunol., 162, 1101-7. Fitzpatrick, F.A. (1991) Inhibition of leukotriene A4 hy- 157. Robbiani, D.F., Finch, R.A., Jager, D., Muller, W.A., drolase/aminopeptidase by captopril. J. Biol. Chem., 266, Sartorelli, A.C. and Randolph, G.J. (2000) The leukotriene 16507-16511. C4 transporter MRP1 regulates CCL19 (MIP-3beta, ELC)- 142. Owman, C., Nilsson, C. and Lolait, S.J. (1996) Cloning of dependent mobilization of dendritic cells to lymph nodes. cDNA encoding a putative chemoattractant receptor. Ge- Cell, 103, 757-68. nomics, 37, 187-94. 158. Rouzer, C.A., Ford-Hutchinson, A.W., Morton, H.E. and 143. Pangburn, M.K. and Walsh, K.A. (1975) Thermolysin and Gillard, J.W. (1990) MK886, a potent and specific leukot- neutral protease: Mechanistic considerations. Biochemis- riene biosynthesis inhibitor blocks and reverses the mem- try, 14, 4050-4054. brane association of 5-lipoxygenase in ionophore- 144. Penning, T.D., Askonas, L.J., Djuric, S.W., Haack, R.A., challenged leukocytes. J. Biol. Chem., 265, 1436-1442. Yu, S.S., Michener, M.L., Krivi, G.G. and Pyla, E.Y. 159. Rouzer, C.A. and Kargman, S. (1988) Translocation of 5- (1995) Kelatorphan and related analogs - potent and selec- lipoxygenase to the membrane in human leukocytes chal- tive inhibitors of leukotriene A4 hydrolase. Bioorg. Med. lenged with ionophore A23187. J. Biol. Chem., 263, Chem. Lett., 5, 2517-2522. 10980-10988. 145. Penning, T.D., Chandrakumar, N.S., Chen, B.B., Chen, 160. Rouzer, C.A., Matsumoto, T. and Samuelsson, B. (1986) H.Y., Desai, B.N., Djuric, S.W., Docter, S.H., Gasiecki, Single protein from human leukocytes possesses 5- A.F., Haack, R.A., Miyashiro, J.M. et al. (2000) Structure- lipoxygenase and leukotriene A4 synthase activites. Proc. activity relationship studies on 1-[2-(4- Natl. Acad. Sci. USA, 83, 857-861. Phenylphenoxy)ethyl]pyrrolidine (SC-22716), a potent 161. Rouzer, C.A. and Samuelsson, B. (1985) On the nature of inhibitor of leukotriene A4 (LTA4) hydrolase. J. Med. the 5-lipoxygenase reaction in human leukocytes: Enzyme Chem., 43, 721-35. purification and requirement for multiple stimulatory fac- 146. Penrose, J.F., Gagnon, L., Goppelt-Struebe, M., Myers, P., tors. Proc. Natl. Acad. Sci. USA, 82, 6040-6044. Lam, B.K., Jack, R.M., Austen, K.F. and Soberman, R.J. 162. Rouzer, C.A. and Samuelsson, B. (1987) Reversible, (1992) Purification of human leukotriene C4 synthase. calcium-dependent membrane association of human leu- Proc. Natl. Acad. Sci. USA, 89, 11603-11606. kocyte 5-lipoxygenase. Proc. Natl. Acad. Sci. USA, 84, 147. Penrose, J.F., Spector, J., Baldasaro, M., Xu, K.Y., Boyce, 7393-7397. J., Arm, J.P., Austen, K.F. and Lam, B.K. (1996) Molecu- 163. Rouzer, C.A., Shimizu, T. and Samuelsson, B. (1985) On lar cloning of the gene for human leukotriene C4 synthase the nature of the 5-lipoxygenase reaction in human leuko- - organization, nucleotide sequence, and chromosomal lo- cytes: Characterization of a membrane-associated stimula- calization to 5q35. J. Biol. Chem., 271, 11356-11361. tory factor. Proc. Natl. Acad. Sci. USA, 82, 7505-7509. 148. Penrose, J.F., Spector, J., Lam, B.K., Friend, D.S., Xu, 164. Rybina, I.V., Liu, H., Gor, Y. and Feinmark, S.J. (1997) K.Y., Jack, R.M. and Austen, K.F. (1995) Purification of Regulation of leukotriene A4 hydrolase activity in endo- human lung leukotriene C4 synthase and preparation of a

32

References

thelial cells by phosphorylation. J Biol Chem, 272, 31865- 180. Strömberg Kull, F. and Haeggström, J.Z. (1998) Purifica- 31871. tion and characterization of leukotriene A4 hydrolase from 165. Samuelsson, B. (1983) Leukotrienes: Mediators of imme- Xenopus laevis oocytes. FEBS Lett., 433, 219-222. diate hypersensitivity reactions and inflammation. Science, 181. Tager, A.M., Dufour, J.H., Goodarzi, K., Bercury, S.D., 220, 568-575. von Andrian, U.H. and Luster, A.D. (2000) BLTR medi- 166. Sanak, M., Pierzchalska, M., Bazan-Socha, S. and Szczek- ates leukotriene B4-induced chemotaxis and adhesion and lik, A. (2000) Enhanced expression of the leukotriene C4 plays a dominant role in accumulation in a synthase due to overactive transcription of an allelic vari- murine model of peritonitis. J. Exp. Med., 192, 439-446. ant associated with aspirin-intolerant asthma. Am. J. Resp. 182. Takasaki, J., Kamohara, M., Matsumoto, M., Saito, T., Cell Mol. Biol., 23, 290-6. Sugimoto, T., Ohishi, T., Ishii, H., Ota, T., Nishikawa, T., 167. Sanak, M., Simon, H.U. and Szczeklik, A. (1997) Leukot- Kawai, Y. et al. (2000) The molecular characterization riene C4 synthase promoter polymorphism and risk of as- and tissue distribution of the human cysteinyl leukotriene pirin-induced asthma. Lancet, 350, 1599-600. CysLT2 receptor. Biochem. Biophys. Res. Commun., 274, 168. Sanger, F., Nicklen, S. and Coulson, A.R. (1977) DNA 316-322. sequencing with chain-terminating inhibitors. Proc. Natl. 183. Thunnissen, M.G.M., Nordlund, P. and Haeggström, J.Z. Acad. Sci. USA, 74, 5463-5467. (2001) Crystal structure of human leukotriene A4 hy- 169. Sarau, H.M., Ames, R.S., Chambers, J., Ellis, C., Elshour- drolase, a bifunctional enzyme in inflammation. Nature bagy, N., Foley, J.J., Schmidt, D.B., Muccitelli, R.M., Str. Biol., 8, 131-135. Jenkins, O., Murdock, P.R. et al. (1999) Identification, 184. Tryselius, Y., Nilsson, N.E., Kotarsky, K., Olde, B. and molecular cloning, expression, and characterization of a Owman, C. (2000) Cloning and characterization of cDNA cysteinyl . Mol. Pharmacol., 56, 657- encoding a novel human leukotriene B4 receptor. Bio- 63. chem. Biophys. Res. Commun., 274, 377-382. 170. Sarkar, G. and Sommer, S.S. (1990) The "megaprimer" 185. Tsai, A., Hsi, L.C., Kulmacz, R.J., Palmer, G. and Smith, method of site-directed mutagenesis. BioTechniques, 8, W.L. (1994) Characterization of the tyrosyl radicals in 404-407. ovine prostaglandin H synthase-1 by isotope replacement 171. Schievella, A.R., Regier, M.K., Smith, W.L. and Lin, L.L. and site-directed mutagenesis. J. Biol. Chem., 269, 5085- (1995) Calcium-mediated translocation of cytosolic phos- 91. pholipase A2 to the nuclear envelope and endoplasmic re- 186. Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., ticulum. J. Biol. Chem., 270, 30749-54. Tashiro, F., Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y. 172. Schultz, M.J., Wijnholds, J., Peppelenbosch, M.P., Ver- et al. (1997) Role of cytosolic phospholipase A2 in aller- voordeldonk, M.J., Speelman, P., van Deventer, S.J., gic response and parturition. Nature, 390, 618-622. Borst, P. and van der Poll, T. (2001) Mice lacking the 187. Wade, M.L., Voelkel, N.F. and Fitzpatrick, F.A. (1995) multidrug resistance protein 1 are resistant to Streptococ- "Suicide" inactivation of prostaglandin I2 synthase: char- cus pneumoniae-induced pneumonia. J. Immunol., 166, acterization of mechanism-based inactivation with isolated 4059-64. enzyme and endothelial cells. Arch. Biochem. Biophys., 173. Scoggan, K.A., Fordhutchinson, A.W. and Nicholson, 321, 453-8. D.W. (1995) Differential activation of leukotriene biosyn- 188. Vallee, B.L. and Auld, D.S. (1990) Zinc coordination, thesis by granulocyte-macrophage colony-stimulating fac- function, and structure of zinc enzymes and other proteins. tor and interleukin-5 in an eosinophilic substrain of hl-60 Biochemistry, 29, 5647-5659. cells. Blood, 86, 3507-3516. 189. Van Sambeek, R., Stevenson, D.D., Baldasaro, M., Lam, 174. Serio, K.J., Hodulik, C.R. and Bigby, T.D. (2000) Sp1 and B.K., Zhao, J.L., Yoshida, S., Yandora, C., Drazen, J.M. Sp3 function as key regulators of leukotriene C4 synthase and Penrose, J.F. (2000) 5 ' Flanking region polymor- gene expression in the monocyte-like cell line, THP-1. phism of the gene encoding leukotriene C4 synthase does Am. J. Resp. Cell Mol. Biol., 23, 234-40. not correlate with the aspirin-intolerant asthma phenotype 175. Shen, R.F. and Tai, H.H. (1986) Immunoaffinity purifica- in the United States. J. Clin. Immunol., 106, 72- tion and characterization of thromboxane synthase from 76. porcine lung. J. Biol. Chem., 261, 11592-9. 190. Welsch, D.J., Creely, D.P., Hauser, S.D., Mathis, K.J., 176. Shimizu, T., Izumi, T., Seyama, Y., Tadokoro, K., Råd- Krivi, G.G. and Isakson, P.C. (1994) Molecular cloning mark, O. and Samuelsson, B. (1986) Characterization of end expression of human leukotriene C4 synthase. Proc. leukotriene A4 synthase from murine mast cells: Evidence Natl. Acad. Sci. USA, 91, 9745-9749. for its identity to arachidonate 5-lipoxygenase. Proc. Natl. 191. Werz, O., Klemm, J., Samuelsson, B. and Radmark, O. Acad. Sci. USA, 83, 4175-4179. (2000) 5-lipoxygenase is phosphorylated by p38 kinase- 177. Smith, W.G., Russell, M.A., Liang, C.-D., Askonas, L.A., dependent MAPKAP kinases. Proc. Natl. Acad. Sci. USA, Kachur, J.F., Kim, S., Souresrafil, N., Price, D.V. and 97, 5261-6. Clapp, N.K. (1996) Pharmacological characterization of 192. Wetterholm, A., Blomster, M. and Haeggström, J.Z. selective inhibitors of leukotriene A4 hydrolase. (1996) Leukotriene A4 hydrolase: a key enzyme in the Prostaglandins Leukot. Essent. Fatty Acids, 55, suppl. 1, biosynthesis of leukotriene B4. In Eicosanoids: From Bio- 13. technology to Therapeutic Applications, (Folco, G., 178. Smith, W.L., DeWitt, D.L. and Garavito, R.M. (2000) Samuelsson, B., Maclouf, J. and Velo, G.P., eds.), pp. 1- Cyclooxygenases: structural, cellular, and molecular biol- 12. Plenum Press, New York. ogy. Ann. Rev. Biochem., 69, 145-82. 193. Wetterholm, A. and Haeggström, J.Z. (1992) Leukotriene 179. Strömberg, F., Hamberg, M., Rosenqvist, U., Dahlén, S.- A4 hydrolase: an anion activated peptidase. Biochim. Bio- E. and Haeggström, J.Z. (1996) Formation of a novel me- phys. Acta, 1123, 275-281. tabolite of leukotriene A4 in tissues of Xenopus laevis. 194. Wetterholm, A., Haeggström, J.Z., Samuelsson, B., Yuan, Eur. J. Biochem., 238, 599-605. W., Munoz, B. and Wong, C.H. (1995) Potent and selec-

33 Martin J. Mueller

tive inhibitors of leukotriene A4 hydrolase - effects on pu- mutations regarding conserved histidine residues. J. Biol. rified enzyme and human polymorphonuclear leukocytes. Chem., 268, 2535-2541. J. Pharmacol. Exp. Ther., 275, 31-37. 209. Zhang, Y.-Y., Rådmark, O. and Samuelsson, B. (1992) 195. Wetterholm, A., Medina, J.F., Rådmark, O., Shapiro, R., Mutagenesis of some conserved residues in human 5- Haeggström, J.Z., Vallee, B.L. and Samuelsson, B. (1991) lipoxygenase: Effects on enzyme activity. Proc. Natl. Recombinant mouse leukotriene A4 hydrolase: a zinc met- Acad. Sci. USA, 89, 485-489. alloenzyme with dual enzymatic activities. Biochim. Bio- 210. Zhao, J.L., Austen, K.F. and Lam, B.K. (2000) Cell- phys. Acta, 1080, 96-102. specific transcription of leukotriene C4 synthase involves 196. Wetterholm, A., Medina, J.F., Rådmark, O., Shapiro, R., a Kruppel-like transcription factor and Sp1. J. Biol. Haeggström, J.Z., Vallee, B.L. and Samuelsson, B. (1992) Chem., 275, 8903-10. Leukotriene A4 hydrolase: Abrogation of the peptidase ac- tivity by mutation of glutamic acid-296. Proc. Natl. Acad. Sci. USA, 89, 9141-9145. 197. Wong, A., Hwang, S.M., Cook, M.N., Hogaboom, G.K. and Crooke, S.T. (1988) Interactions of 5-lipoxygenase with membranes: Studies on the association of soluble en- zyme with membranes and alterations in enzyme activity. Biochemistry, 27, 6763-6769. 198. Woods, J.W., Coffey, M.J., Brock, T.G., Singer, I.I. and Peters-Golden, M. (1995) 5-lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation. J. Clin. Invest., 95, 2035-2046. 199. Woods, J.W., Evans, J.F., Ethier, D., Scott, S., Vickers, P.J., Hearn, L., Heibein, J.A., Charleson, S. and Singer, I.I. (1993) 5-lipoxygenase and 5-lipoxygenase activating protein are localized in the nuclear envelope of activated human leukocytes. J. Exp. Med., 178, 1935-1946. 200. Wu, G., Wei, C., Kulmacz, R.J., Osawa, Y. and Tsai, A.L. (1999) A mechanistic study of self-inactivation of the per- oxidase activity in prostaglandin H synthase-1. J. Biol. Chem., 274, 9231-7. 201. Xu, G.Y., McDonagh, T., Yu, H.A., Nalefski, E.A., Clark, J.D. and Cumming, D.A. (1998) Solution structure and membrane interactions of the C2 domain of cytosolic phospholipase A2. J. Mol. Biol., 280, 485-500. 202. Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y. and Shimizu, T. (1997) A G-protein-coupled receptor for leu- kotriene B4 that mediates chemotaxis. Nature, 387, 620- 624. 203. Yokomizo, T., Kato, K., Hagiya, H., Izumi, T. and Shi- mizu, T. (2001) Hydroxyeicosanoids bind to and activate the low affinity leukotriene B4 receptor, BLT2. J. Biol. Chem., 276, 12454-9. 204. Yokomizo, T., Kato, K., Terawaki, K., Izumi, T. and Shimizu, T. (2000) A second leukotriene B4 receptor, BLT2: A new therapeutic target in inflammation and im- munological disorders. J. Exp. Med., 192, 421-431. 205. Yuan, J.H., Birkmeier, J., Yang, D.C., Hribar, J.D., Liu, N., Bible, R., Hajdu, E., Rock, M. and Schoenhard, G. (1996) Isolation and identification of metabolites of leu- kotriene A4 hydrolase inhibitor SC-57461 in rats. Drug Metab. Dispos., 24, 1124-1133. 206. Yuan, W., Wong, C.-H., Haeggström, J.Z., Wetterholm, A. and Samuelsson, B. (1992) Novel tight-binding inhibi- tors of leukotriene A4 hydrolase. J. Am. Chem. Soc., 114, 6552-6553. 207. Zaitsu, M., Hamasaki, Y., Matsuo, M., Kukita, A., Tsuji, K., Miyazaki, M., Hayasaki, R., Muro, E., Yamamoto, S., Kobayashi, I. et al. (2000) New induction of leukotriene A(4) hydrolase by interleukin-4 and interleukin-13 in hu- man polymorphonuclear leukocytes. Blood, 96, 601-9. 208. Zhang, Y.-Y., Lind, B., Rådmark, O. and Samuelsson, B. (1993) Iron content of human 5-lipoxygenase, effects of

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