Biochimica et Biophysica Acta, 1080( 1991) 96-102 !~ 1991 Elsevier Science Publishers B.V. All rights reserved 0167-4838/91/$03 51) ADONIS 016748389100308L
96
BBAPRO 34032
Recombinant mouse leukotriene A 4 hydrolase: a zinc metalloenzyme with dual enzymatic activities Anders Wetterholm ~, Juan F. Medina ~, Olof RSdmark ~, Robert Shapiro 2, Je%,~, Z HaeggstriSm ~, Bert L. Vallee 2 and Bengt Samuelsson i I Department of Physiological Chemistry, Karolinska lnstitutet, Stockhobn (Sweden) and 2 Ce, ,terror Biochemt~al and Btophysieal Sciences and Medicine, Harcard Medical School, Boston, MA (U.S.A.)
(Received 27 M ~rch 1991)
Key words: Leukotriene A4; Hydrolase: Metalloenzyme
Recombinant mouse ieukotriene A 4 hydrolase was expressed in Escherichia coli as a fusion protein w i t h t e n additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of z i n c / m o l of enzyme. Apparent kinetic c o n s t a n t s ,tK m and Vw~) for the conversion of leukotriene A 4 to leukotriene B 4 (at 0~C, pH 8) were 5 /zM and 900 n m o l / m g per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine° 4-nitroanilide. K . and Vmx for this reaction (at 37°C, pH 8) were 680 p M and 365 n m o l / m g per min, respectively. A p o - l e u k o t r i e n e A 4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A 4 resulted in a dose-dependent inactivation of both enzyme activities.
Introduction Leukotriene A 4 (ETA 4) hydrolase (EC 3.3.2.6) catalyzes the hydrolysis of the ailylic epoxide LTA4 (5(S)t r a n s - 5 , 6 - o x i d o - 7 , 9 - t r a n s - 1 1 , 1 4 - c i s - e icosate tra e no ic acid) into the proinflammatory substance LTB4 (5S, 12R-dihydroxy-6,14-cis-8, l O - t r a n s - e i c o s a t e t r a e n o i c acid) (for review see Ref. 1). LTA 4 hydrolase has been purified from a variety of tissues and species as a soluble, monomerie protein with a molecular weight of approx. 69000 (for reviews see Refs. 2 and 3). The
Abbreviations: LTA4, leukotriene A 4, 5(S)-trans-5,6-oxido-7,9-trans11,14-c/s-eicosatetraenoic acid; L T B 4, leukotriene B4, 5(S),12(R)-dihydroxyo6,14-eis-8,!O-trans-eicosatetraenoic acid: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Bistris. bis(2-hydroxyethyl)iminotris(hydro~methyl)methane: RP-HPLC, reverse-phase high-performance liquid chromatography; FPLC. fastprotein liquid chromatography; IPTG, isopropyl-fl-D-thiogalactopyranoside. Correspondence: A. Wenerholm, Department of Phys,t,~ogtc~l Chemistry, Karolinska lnstitutet, Box 60400, S. il~' 01 Stockholm,
Sweden.
human enzyme has been cloned from placenta and spleen c D N A libraries and sequenc~:d [4,5]. L T A 4 hydrolase is widely distributed, and is expressed also in cells apparently devoid of 5-1ipoxygenase/LTA 4 synthase activity [6-8]. Very little is known about the catalytic mechanism of LTA 4 hydrolase, but recently a sequence similarity between LTA 4 hydrolase and certain zinc metalloenzymes, for instance, aminopeptidase M and thelmolysin, was demonstrated, revealing the presence of a zinc binding motif in the enzyme [9,10]. Accordingly, human LTA 4 hydrolase was found to contain one atom of zinc per enzyme molecule [11,12]. In addition, the enzyme was recently shown to exhibit peptidase activity towards synthetic aromatic amides [12-14]. Mouse spleen LTA 4 hyd~ olase was recently cloned, sequenced and expressed in E. coli as an active fusion protein [15]. For studies on the effects of site directed mutagenesis on catalytic properties and metal content, we developed a rapid and reproducible purification procedure for recombinant mouse L T A 4 hydrolase. Physical and kinetic properties of the enzyme, including the zinc content and peptidase activity, are described.
97 Materials and Methods
LTA 4 methyl ester (Mcrck-Frosst, Canada) was saponified in tetrahydrofuran with 1 M LiOH (6% v/v) for 48 h at 4°C. ZnSO 4 and CoCI 2 (Johnson-Matthey specpurc) were dissolved in reagent grade Milli-Q water (Waters-Millipore). Alanine-4-nitroanilide, leucine4-nitroanilidc and i,i6-pheeanthroline were from Sigma. 4-Nitroaniline was from Kebo, Stockholm.
Enzyme purification Cell preparation and protein precipitations
Pharmacia). The column was equilibrated with 20 mM Tris-HCI (pH 8), containing 20% ammonium sulphate. Adsorbed proteins were eluted with a gradient from 20-0% ammonium sulphate. Active fractions were pooled and the buffer was changed to 25 mM Bistris (pH 7.1), by repeated centrifugation on a Ce,~tricon-30 microconcentrator (Amicon). For the final purification, a chromatofocusing column, Mono-P (5 x 200 mm, Pharmacia), was used. The starting buffer was 25 mM Bistris (pH 7.1), followed by 10% Polybuffer 74 (pH 4, pH adjusted with saturated iminodiacetic acid), at a flow rate of 0.5 ml/min. A peak exhibiting LTA 4 hydrolase activity was eh,ted at pH ( = p l ) 5.5. Th~ buffer was changed to 10 mM Tris-HCI (pH 8), by ultrafiltration as above, and aliquots of the purified enzyme were lyophilizcd and stored at -20°C. SDS-PAGE was performed on a Phast system (Pharmacia) using 10-15% gradient gels. The p l of the purified protein was determined by isoelectric focusing on a Phast IEF 4-6.5 gel. Bands of protein were visualized by staining with Coomassie brilliant blue. Protein concentrations were determined by the Bradlord method [16], using bovine serum albumin as standard. Zinc determinations were performed by atomic absorption spectrometry on a Perkin-Elmer Model 5000 electrothermal atomic absorption instrument. The result is an average of triplicate determinations on different dilutions of sample. Protein concentrations were determined by amino acid analysis.
Recombinant mouse LTA 4 hydrolase, a fusion protein with the first ten amino acids of the fl-galactosidase gene, was purified from E. coli (JM 101) transformed with the plasmid pULTA 4, the cDNA of which was cloned from a mouse splccn library [15]. Ceils were cultured at 37°C in M9 medium containing 0.2% casamino acids, 0.4% glucose, 2 mM MgSO4 and 75 # g / m l of ampicillin. IPTG (0.5 mM) was added at A~a~ ,,, ~ 0.2 and the incubation was allowed to continue for 2-3 h. Cells from 1 to 3 liter cultures were harvested by centrifugation (1000×g, 10 rain) and resuspended in 25 ml 50 mM Tris-HCl, (pH 8) supplemented with EDTA (5 mM), dithiothreitol (2 mM) phenyimethylsuifonyl fluoride (1 mM) and soybean trypsin inhibitor (60/.tg/ml), referred to as buffer A. All further mar~ipulations were carried out c,n ice or in a cold cabinet. Homogenization of cells was performed by sonication (4 x 15 s) using a Branson Sonifier, model S-125, at setting 4. The homogenate was centrifuged at 1 0 0 0 0 x g for 15 min and streptomycin sulphate (5% v / v of a 10% w / v solution) was added to the supernatant. Precipitated material was removed by centrifugation (10000 x g) and solid ammonium sulphate was added gradually to 40% saturation. Following another centrifugation, ammonium sulphate was added to 80% saturation and the resulting precipitate was collected by centrifugation. The 10000 X g pellet was resuspended in buffer A and subjected to ultrafiltration, using an Amicon PM-30 filter (Danvers MA), for buffer change (repeated three times).
Apo-LTA 4 hydrolase was prepared by treating the purified enzyme (0.5-1 mg, 1 mg / m l ) with 1,10phenanthroline (I0 mM) in 50 mM Hepes (pH 8), for 24-48 h at 4°C. The chelator was subsequently removed by ultrafiltration, as judged by the disappearance of absorbance at 264 nm [17]. The apoenzyme was stored in 10 mM Tris-HCl (pH 8) at 4°C and was stable for at least 2 weeks. For reconstitution with zinc or cobalt, the apoenzyme was treated with metal salt dissolved in 10 t~l Milli-Q water for 30 min at room temperature prior to determination of enzymatic activity.
Chromatographies, electrophoresis and zinc analysis
Determinations of enzyme activities
An anion exchange column (Mono-Q, 10 x 100 mm, Pharmacia) was equilibrated with 10 mM Tris-HCI (pH 8), at a flow rate of 3 m l / m i n and the protein obtained from precipitations was applied to the column. After elution of nonadsorbed material, a linear gradient of KCI was star~ed. Protein with LTA 4 hydrolase activity eluted between 0.10-0.15 M KCI. The Mono-Q pool was supplemented with 20% (w/v) ammonium sulphate and subjected to hydrophobic interaction chromatography on a Phenyl-Superose column, (5 x 50 mm,
The LTA 4 hydrolase activity was determined by incubating the enzyme (2-3 /~g in 100 #1 10 mM Tris-HCl, pH 8) with LTA 4 in 1 /.tl tetrahydrofuran (4-0 nmol). LTA 4 is a very labile comp~Jt,nd, which rapidly decomposes at high temperature and acidic pH. In order to reduce the influence of substrate instability in kinetic experiments, short-time incubations were performed on ice for 15 s. The reaction was quenched with 200 ttl of methanol and a defined amount of internal standard, prostaglandin B I (Upjohn), was
Preparation o1"apoenzyme
98 added. The samples were acidified to pH 3 with dilute HCI immediately bclorc RP-HPI,C on a column (Ultrosphere ODS. 250 × 4.6 ram) eluted with a mixture of a c e t o n i t r i l e / m e t h a n o l / w a t e r / a c e t i c acid (30: 35:35:0.01, v / v ) at a flow rate of 1 m l / m i n . The absorbance of the eluate (270 nm) was m o n i t o r e d continuously. Quantitations of LTB 4 were made by measurements of peak height ratios between LTB 4 and prostaglandin B 1, as described [18]. The peptidase activity was d e t e r m i n e d spectrophotometrically, essentially as described [19]. T h e substrates were dissolved in 50 m M Tris-HCI (pH 8) to a coucentration of 1 mM. Following addition of enzyme ( 1 - 4 #g). the product (4-nitroaniline) was m e a s u r e d as the increase in absorbanee at 410 nm, assuming an extinction coefficient of 8850 M - ~ c m - m. Alternatively, the assay was performed in the wells of a microtiterplate, and the absorbance at 405 nm was m e a s u r e d using a multiscan s p e c t r o p h o t o m e t e r , M C C / 3 4 0 (Labsystems). Quantitations were made from a standard curve obtained with known amounts of 4nitroaniline in 50 mM Tris-HCI (pH 8). Correction for spontaneous hydrolysis of the substrate was m a d e by subtracting the absorbance of control incubations without enzyme. Time-courses for the two enzymatic activities and effects of repetitive additions of LTA4 on the formation of LTB 4 and 4-nitroaniline were studied in the following way: LTA 4 hydrolase ( 2 5 / x g in 1 ml 10 mM Tris-HCI, p H 8) was kept on ice a n d an aliquot was removed for assay of peptidase activity, by incubation with alanine-4-nitroanilide. To the remaining solution, LTA 4 was added ( 8 0 / ~ M ) and aliquots were q u e n c h e d with methanol at the desired time points. After 5 min, an additional sample was removed for incubation with alanine-4-nitroanilide. A second dose of LTA,~ (80 # M , final concentration) was then a d d e d and the assays of LTB 4 formation and peptidase activity were repeated.
Mr (kD)
2
1
3
4
6
94.0 67,0
.....
43.0
30.0 20.1 14.4 Fig I. 5DS-PAGE of LTA 4 hydrolase at different steps of purificalion. Eleetrophoresis of the samples was carried out on a PhastGradient gel 10-15 (Pharmacia Phast System) and stained with Coomassie brilliant blue. The molecular mass markers were; phosphorylase b (94.(I kDa), bovine serum albumin (67.(I kDa), ovalbumin (43.0 kDa), carbonic anhydrase (30.0 kDak soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). The gel shows molecular weight markers (lane I), 100110xg supernatant (lane 21, 40-80r~ ammonium sulfate precipitate (lane 3), fractions from Mono-O (lane 4), PhenyI-Superose (lane 5) and Mono-P (hmc 6) chromatographies.
used as the final purification step (Fig. 3) a n d also by isoelectric focusing on a Phast I E F gel (data not shown). Four differen t enzyme batches were subjected to atomic absorption spectrometry which revealed the p r e s e n c e of 0.94, 0.66, 0.76 a n d 1.22 g-atom of zinc p e r mol of enzyme (Table 11). T h e m e a n value of these d e t e r m i n a t i o n s is 0.90 g - a t o m / m o l a n d since the theoretical value should bc an integer, tile closest approximation is one zinc atom p e r enzyme molecule.
(Nlla)2St)4 -20%
....
b
--....
Results
400-
/)l/ 3oot . l OOy
Purification and chemical properties of the enzyme R e c o m b i n a n t mouse L T A 4 hydrolase was purified to a p p a r e n t homogeneity, as judged by S D S - P A G E (Fig. 11. T h e procedure involved streptomycin a n d amm o n i u m sulphate precipitations, anion exchmJge, hydrophobic interaction (Fig. 2) and chromatofocusing chromatographies (Fig. 3) on FPLC. T h e enzyme was purified 19-fold with a yield of 60% (Table I). F r o m 1 I of cell culture, 1 - 2 mg of pure enzyme could be obtained. T h e molecular weight of the et,zyme was estimated to 69000 from S D S - P A G E (theoretical value 69860). The p l was d e t e r m i n e d to 5.5 by chromatofocusing,
5
- -
t
l0
I
l
20 Volume (ml) Fig. 2. PhenyI-Superose chromatography of mouse LTAa hydrolase. Enzymatically active fractions from Mono-O chromatography were pooled and 20% (w/v) solid ammonium sulfate was added. The column, PhenyI-Superose (5 x 50 mm), was equilibrated with 20 mM Tris-HCI, pH 8 with 20% (NH4)2 SO4 at a flow rate of 0.7 ml/min. After sample loading, a linear gradient of 20-0% ammonium sulfate was started. Aliquots (2 p,I) of the fractions were diluted to 100 p.I with 10 mM Tris-HCI, oH 8 and incubated with LTA 4 (35/zM) for 1 min at room temperature. The enzymatic product, LTB.;, was quantirated by RP-HPLC (see M~terials and Methods).
99 TABLE I Purification of recombinant mouse L 744 hvdrohtsc
LTA 4 hydrolase from 3 I cell culture was purified as dcscribed in Materials and Methods. A!iq'aols of the enzyme, afler indicated purification steps, were incubated with LTA 4 (45 /aM final c(mcenlration) for I rain at room Icmperature. Formation of [.TB 4 was analyzed by RP-IIPLC Fraction 1000(1× g-supernatan! Precipitations Mono-Q PhenyI-Superose Mono-P
Volume
Total protein
(ml)
(m~)
30
162 126 35 12.5 5.2
61)
5 5 2
Total activity (nmol/min/
I600 I 018 ~62
pH
7~\ .... IO(tO
--..
>
-5 750
g c
--
-4
{
Yield
Pu:ification
(r4)
(-fl)ld)
1011 87 78 64 60
I 3,5 S
10 II 3t~ 81
I 4110 I 256
Catalytic properties, thne-cour~es attd L T A 4 -mediated hlactit'ation A p p a r e n t v a l u e s o f K m a n d 1/",.... for t h e c p o x i d e h y d r o l a s e activity o f r e c o m b i n a n t m o u s e L T A 4 h y d r o lase w e r e d e t e r m i n e d f r o m i n c u b a t i o n s o f t h e e n z y m e (3 lag) with L T A 4 ( 5 - 1 0 0 taM'b for 15 s o n ice. T h e rate o f ~' l t..vl , , 4 p. .l g. u. u a' ~.t l.t.m. -__ at t h e d i f f e r e n t su', q r a t c c o n c e n t r a t i o n s w e r e e x t r a p o l a t e d to I m i n a n d t h e d a t a plott e d a c c o r d i n g to H a n e s . F r o m this plot K m a n d V, .... w e r e c a l c u l a t e d to 5 # M a n d 9(X) n m o l / m g p e r m i n , respectively (data not shown). T h e e n z y m e w a s a l s o f o u n d to exhibit p e p t i d a s e activity t o w a r d s s y n t h e t i c a m i d e s . T h e r e a c t i o n rate w a s c o n s t a n t for s e v e r a l h o u r s , w i t h o u t a n y sign o f i n a c t i v a t i o n (cf. Fig. 4B). T h e specific activity, u s i n g a l a n i n e - 4 - n i t r o a n i l i d c (i r a M ) as s u b s t r a t e , w a s calculated to 270 n m o l / m g p e r m i n at 37°C. A n o t h e r a m i n o acid c o n j u g a t e , l c u c i n c - 4 - n i t r o a n i l i d e , w a s also a s u b -
Specific actixit.~ (nmol/mg × rain)
185
;9
s t r a t e for m o u s e L T A 4 h y d r o l a s c , with a specific activity a p p r o x . 711% o f that o b t a i n e d with a l a n i n e - 4 - n i t r o anilide. A p p a r e n t K m a n d V,,~,X for t h e hydrolysis o f a l a n i n e - 4 - n i t r o a n i l i d e (at 37°C, p H 8) w e r e e s t i m a t e d f r o m H a n e s ' plot to 680 # M a n d 365 n m o l / m g p c min, r e s p e c t i v e l y ( d a t a not s h o w n ) . T h e i n c r e a s e in a b s o r b a n c e at 405 n m w a s r e c o r d e d d u r i n g a 30 m i n p e r i o d f r o m i n c u b a t i o n s o f e n z y m e (1 p.g) with s u b strate (0.06-1 mM). T h e t i m e - c o u r s e s a n d e f f e c t s o f repetitive a d d i t i o n s o f L T A 4 o n t h e two e n z y m a t i c activities a r e s h o w n in Fig. 4 A a n d B. T h e a m o u n t s o f L T B 4 i n c r e a s e d r a p i d l y a n d a l m o s t l i n e a r with t i m e d u r i n g t h e first 30 s a n d t h e n g r a d u a l l y levelled off. w h i c h in p a r t w o u l d be e x p e c t e d f r o m t h e instability o f t h e s u b s t r a t e w h i c h h a s a half-life at 4°C o f a b o u t 30 s [20]. F o l l o w i n g a s e c o n d d o s e o f L T A 4, o n l y s m a l l a d d i t i o n a l a m o u n t s o f L T B 4 w e r e p r o d u c e d (Fig. 4A). In this e x p e r i m e n t , also t h e p e p t i d a s e activity o f L T A 4 h.¢drolase w a s d e t e r m i n e d for aliqttots r e m o v e d b e f o r e a n d a f t e r a d d i t i o n s o f L T A 4 (Fig. 4B). F o r e a c h aliquot, t h e p e p t i d a s e activity w a s l i n e a r with t i m e for m o r e t h a n 2 h, w i t h o u t a n y sign o f i~metivation. H o w e v e r , in e n z y m e a l i q u o t s rem o v e d a f t e r a d d i t i o n s o f L T A 4, t h e p e p t i d a s e activities w e r e d e c r e a s e d ( a p p r o x . 5 0 % for e a c h d o s e o f L T A 4 ) , i n d i c a t i n g t h a t e x p o s u r e o f t h e e n z y m e to L T A 4 also a f f e c t e d s u b s e q u e n t p e p t i d a s e activity.
500
=
.
" ~
750
.,
500
~
E 0
0.0
I
I
I
I
0.5
1.0
1.5
2,0
Molar ratio metal/enzyme Fig. 5. Effects of zinc and cobalt on the peptidase activity of apo-LTA 4 hydrolase. APO-LTA4 hydrolase (3 /zg in 5 /~1 10 mM Tris-HCI, pH 8) was preincubated with increasing amounts of zinc (o) or cobalt (e) (10/J.I) in the wells of a microtiter plate After 30 rain, alanine-4-nitroanilide (1 mM in 50 mM Tris-HCI, pH 8. 235 #1) was added and the formation of 4-nitroaniline was recorded spectrophotometrically at 405 nm. Specific activities were calculated from 30 rain incubations at 22°C and represent means of duplicate samples.
E
N
¢ t~ +
0 r,..) +
0
Fig, ~. Specific epoxide hydrolase activities of apoenzyme, holoenzyme and apoenzyme reconstituted with zinc or cobalt. Apo-LTA 4 hydrolase (3 ~g)was preincubated with one equivalent of the desired metal in 100 ~l 10 mM Tris-HCI (pH 8) for 30 rain. Duplicate samples (3 /~g) of untreated apoenzyme, holoenzyme and reconstituted apoen~me were incubated with LTA 4 (80 p,M) for 15 s at 22°C for determination of the specific activities.
101
lasc. The enzyme was expressed irl E. coli and purified to apparent homogeneity by me;ms of precipitations and three different chromatograpifies on FPLC. with a yield of about 60%. The molecular weight was estimated to approx. 69000 and the p / w a s 5.5. With respect to the epoxide substrate LTA 4, apparent K m and V,.... were 5 u M and 900 n m o l / m g per min, (pH 8, 0°C), and thc time-course of the reaction was characterized by a rapid burst of catalysis, followed by reduced enzymatic activity upon a second dose of substrate. Thus, these physical and kinetic characteristics of recombinant mouse LTA4 hydrolase are similar to those reported for enzymes purified from other sources [3], with thc exception of guinea pig liver E T A 4 hydrolase, which exhibited a ~, .... significantly higher than the others
[21]. An additional enzymatic activity, i.e. hydroly;s of two synthetic amino acid dc,i~,ativcs (alaninc- and leucine-4-nitroanilide)was establ,shed t(~r mouse L] A4 hydrolase. Apparent K m and Vm,, (37°C, 50 mM TrisHCI, 13H 8) of the reactit~r, "~crc ~ctclmined to 680 ~M and 365 nmol/mg per rain, respectively, using alaninc4-nitroanilide as substrate. At a substrate concentration of 1 raM, the specific activity of this reaction was calculated to 270 nmol/mg per min, which can be compared to 380 nmol/mg per min obtained with human leukocyte LTA 4 hydrolase under the same conditions [13]. The recombinant mouse enzyme was shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The zinc atom was necessary for catalytic activity, since the apoenzyme was virtually devoid of both epoxide hydrolase and peptidase activity (Figs, 5 and 6), but could regain its enzymatic activities, towards both substrates, by addition of zinc or cobalt at a 1 : ! molar ratio. Therefore it seems likely that the two activities are exerted via closely related active sites. However, some differences between the two activities were also observed. Thus, enzyme containing cobalt exhibited a higher peptidase activity than the zinc enzyme (while the epoxide hydrolase activity was equal in both cases), and a 2-3 molar excess of zinc slightly inhibited the peptidase, but not the epoxide hydrolase activity. The putative zinc binding site in LTA 4 hydrolase has a very high similarity to the corresponding primary structure of certain other zinc hydrolases, for instance thermolysin, rat, E. coli and human aminopeptidase M, rat enkefalinase, P. aeruginosa elastase and human angiotensin converting enzyme [9,10, 22-26]. In a catalytic zinc site, the metal is coordinated in a tridentate complex to any three out of four amino acid residues from His, Cys, Glu and Asp. The first two ligands (L1 and L2), separated by a 'short spacer' of 1-3 amino acids, are believed to anchor the metal to the protein.
These primary ligands are separated from the third (L3) by a qong spaccr' of 21)-12(I amino acids [10. 27-29]. The fourth ligand is an activated t! !O molecule, which probably is involved in the catalytic reaction [29]. in accordance with these sequence homologies, the zinc atom in human, as well as mouse, LTAa hydrolasc would be coordinated to His-295, His-299 and Glu-318 [ I(1.11,15]. Interestingly, the zinc binding motif of LTA 4 hydrolase is located in the middle of a region of 108 amino acids which are I(X)% conserved between m o u ~ and man, implicating its importance for the enzyme function [15]. LTA 4 hydrolase has previously been shown to bc inactivated by its substrate, possibly by covalent binding of LTA 4 to the enzyme [3tL31]. Recently it was published that this suicide inactivation of LTA 4 hydrolase is intimately related to catalysis, and probably involves the active site [321. The peptidase activity was constant tot more than 2 h, without any sign of inactivation, while exposure of the enzyme to LTA 4, resulted in a dose-dependent decrease of both enzyme activities, although the epoxide hydrolase activity appeared to be more sensitive. Thus. the inhibitory effect of LTA4 on both enzyme activities also suggests that the corresponding catalytic sites are closely associated. The identification of LTA 4 hydrolase as a zinc metalloenzyme with peptidase activity will probably facilitate the development of drugs interfering with biosynthesis of LTB 4 and indeed, the peptidase inhibitor bestatin, and the angiotensin converting enzyme inhibitor captopril, were recently shown to inhibit both enzyme activities of LTA 4 hydrolase [14]. Other peptidases have been shown to take part in, for instance, degradation of peptides in the intestine [33]. processing of neurotransmittors [34], cleavage of angiotensin 11 to angiotensin II1 [35], and conversion of LTC 4 to LTE 4 [36]. The biological role of the peptidase activitx of LTA 4 hydrolase is presently unknown but the enzyme may have diverse functions, depending t~i its location and substrate availability.
Ackpowledgments The authors are greatly indebted to Ms. Eva Ohlson for excellent technical assistance. We also thank Dr. A.W. Ford-Hutchinson (Merck-Frosst, Canada) for the generous gift of ieukotriene A 4. This project was financially supported by the Swedish Medical Research Council (03X-217 and 03X-7467), Gustav V:s 80 ~rsfond, and O.E. and Edla Johanssons foundation.
References 1 Samuelsson,B. (1983)Science220, 568-573. 2 Samuelsson.B. and Funk,C.D. (1989)J. Biol.Chem. 264. 1946919472.
1(12 3 R~dmark, (). ~md I I~lcgg~,lriim..I. 1191~01 Ad'.. Pr~,~laglauldin, Thromboxane ;tnd l,enk~tri~ne Res. 2 0 35-.J2 4 Funk. C.D., Rfidmark, O., Fu. J.Y., Matsumolo, T., J/irnvall. ti., Shimizu. T. and Samnclsson, B. 111187) Pr,~c. Nail. Acad. Sci. USA 84. t~677-6681. 5 Minami. M., Ohno, S.. Kawasaki. II.. R~dml.rk, O.. S;m~nelsson, B.. J~irnvall. tt., Shimizu, T., Seyama. Y. and SuzukL K. ( 19.",71J. Biol. ('hem. 2~2, I "~873-13876. 6 Fitzpatrick, F.A., Liggett, W., Me(lee. ,L. Bunting. S.. Morton. D. and Samuelsstm, B. (19841 J. Biol, Chem. 259, 114(13-114117. 7 Claesson. |t.-E. and Haeggstriim. J. (1988) Eur. J. Biochem. 173. 93- IIl~}. 8 Medina. J.F, Barrios. C., Funk. C D , . Larsson. O,. Haeggstriim, J. an,I Rfidmark, O. (199(11 Eur. J. Hiochem. It/I. 27 ~31, 9 Malfioy. B.. Kado-, ong, 11., Gros. t'., (;ir~r,. B. Schwartz, J.-(.. and Hellmiss. R. (1989) Bfi~hem Biophys. Res. ('~mmn,~. 161. 23h-241. I(I Vallee, B.L. and Auld, D.S. (1991)1 Bitvchemistry_ 29, 5h47 5651~ I I Itaeggstriim. J.Z.. Wetlerholm. A., Shapiro. R., Vallee. B.L. and Samuelsson, B, (19~)1 Biochem Biophys. Res. Commun. 172. 965-9711. 12 Minami. M.. Ohishi, N., Muloh. I1.. lzumi. T.. Bito. H., Wada, It., Seyama, ~.. Toh, H and Shimizu. T. 1199111 Biochem. Biophys. Res. ('ommun. i73. 6211-62~. 1~ ftacggstriim. J . Z , Wetterholm. A., Vallee. B i . and Samuelsson. B. (1991)) Biochem. Biophys. Res. Commun. 173.431-437. 14 ('irning, L.. Krivi. G. and Fitzpatrick. F.A. 119911 J. Biol. C h e m 266. 1375-1378. 15 Medina. J.F., Rfidmark, O., Funk, C.D. and Haeggstr6m. J.Z. (1991) Biochem. Biophys, Res. Commun. 175. 1516-1524, 16 Bradford, M.M. (19761 Anal. Bi~chem. 72. 248-254. 17 Wagner, F.W. 11988) Methods Enzymol 158, 21-32. 18 Rfidmark, O.. Shimizu, T.. J6rnvalt, It. and Samuelsson, B. (19841 J. Biol. Chem. 259, 12339-12345. 19 Sj6str/im, H.. North. O., Jeppesen, L., Staun, M.. Svensson. B. and Christian,sen, L. (19781 Eur. J. Biochem. 88. 5113-511. 211 Fitzpatrick. F.A., Morton, D.R. and Wynalda. M.A. 119821 J. Biol. Chem. 257, 46811-4683.
21 Ftacggstr6m. J., Bcrgman. T., Jiirnvall, tt, and Rfidmark. O. 119881 Eur. J. Biochem t74, 717-724. 22 Olscn, J., (owcll. G.M.. Konigsi~ofcl, E,, Danielsen, E M . . Moiler. J.. Laustsen. L,. ttansen, O.C., Welinder. K.G., Engberg, J., tlunziker. W., Spies,s, M., Sj6slriim, H. and Nor~n. O. 119881 FFH';. l e t t "~3~, 3117-314. 23 Foghno, M., Gharbi. S, and Lazdunski, A. (19861 Gene 49, 3113 3119. 24 Beret, R.A. and Iglewski, B.It. 11988) J. Bacteriol. 1711. 43094314. 25 Malfroy. B., Schofield. P.R,, Kuang, W.-J.. Seebung, P.II. Mason, A.J. and Itenzel, W.J. (19871 Bit~hem. Biophys. Res. ~k~mmun. 144, 59 -~6. 26 Stmbrier, F., Alhenc-Gelas. F.. Hubert, C., Allegrini. J.. John, M.. Tregear, G. and (k)rvol, P. 119881 Proc. Natl. Acad. Sci. USA 85. 9386~931~1 "7 Vallee, H.L. anti Auld. D.S. (19911 in Melhod~ in Protein Sequence Analysis (J&nvall. H. and Ihi6g, J.-O., ed~.), pp. 363-372 Birkhatnscr Verlag, qasel. Switzerland. 28 Vallec. B.L. and Auld. D.S. 119891 FEBS Letl. 257. 138~-1411. 29 Vallcc. B,L. and Auld. D.S (199111 Proc. Natl. Acad. Sci. USA 87. 220-224, 3() Evans. J.F.. Nathaniel. D.J.. Zamh~mi. R J . and Ford-Hulchinson, A.W, ( 19851 J. Biol. Chem. 2011, 111960-1119711. 31 McGee. J. and Fitzpatrick, F.A. 119851 J. Biol. ('hem. 261;, 12832-12~",37. 32 ()rning, l,., Jones, D.A. and Fitzpatrick. F.A. (Ig911) |. Biol. Chem. 265. 14911 - 14916. 33 Maroux, S.. Lxmvard, D. and Baratti. J. 119731 Biochim. Biophys. Acta 321,282-295. 34 Lynch, D.R. and Snyder, S.H. 119861 Annu. Rev. Biochem. 55. 773-799. 35 Reid. I.A., Morris. B.J. and Ganong. W.F. 119781 Ann. Rev. Phy,siol. 411, 377-411) 36 Sok, D.-E., Pal. J.-K.. Attache, V. ,rod Sih. C.J. 119801 Proc. Natl. Acad. Sci, USA 77, 6481-6485.
96
BBAPRO 34032
Recombinant mouse leukotriene A 4 hydrolase: a zinc metalloenzyme with dual enzymatic activities Anders Wetterholm ~, Juan F. Medina ~, Olof RSdmark ~, Robert Shapiro 2, Je%,~, Z HaeggstriSm ~, Bert L. Vallee 2 and Bengt Samuelsson i I Department of Physiological Chemistry, Karolinska lnstitutet, Stockhobn (Sweden) and 2 Ce, ,terror Biochemt~al and Btophysieal Sciences and Medicine, Harcard Medical School, Boston, MA (U.S.A.)
(Received 27 M ~rch 1991)
Key words: Leukotriene A4; Hydrolase: Metalloenzyme
Recombinant mouse ieukotriene A 4 hydrolase was expressed in Escherichia coli as a fusion protein w i t h t e n additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was shown to contain one mol of z i n c / m o l of enzyme. Apparent kinetic c o n s t a n t s ,tK m and Vw~) for the conversion of leukotriene A 4 to leukotriene B 4 (at 0~C, pH 8) were 5 /zM and 900 n m o l / m g per min, respectively. The purified enzyme also exhibited significant peptidase activity towards the synthetic amide alanine° 4-nitroanilide. K . and Vmx for this reaction (at 37°C, pH 8) were 680 p M and 365 n m o l / m g per min, respectively. A p o - l e u k o t r i e n e A 4 hydrolase, prepared by treating the enzyme with 1,10-phenanthroline, was virtually inactive with respect to both enzymatic activities, but could be reactivated by addition of stoichiometric amounts of zinc or cobalt. Exposure of the enzyme to leukotriene A 4 resulted in a dose-dependent inactivation of both enzyme activities.
Introduction Leukotriene A 4 (ETA 4) hydrolase (EC 3.3.2.6) catalyzes the hydrolysis of the ailylic epoxide LTA4 (5(S)t r a n s - 5 , 6 - o x i d o - 7 , 9 - t r a n s - 1 1 , 1 4 - c i s - e icosate tra e no ic acid) into the proinflammatory substance LTB4 (5S, 12R-dihydroxy-6,14-cis-8, l O - t r a n s - e i c o s a t e t r a e n o i c acid) (for review see Ref. 1). LTA 4 hydrolase has been purified from a variety of tissues and species as a soluble, monomerie protein with a molecular weight of approx. 69000 (for reviews see Refs. 2 and 3). The
Abbreviations: LTA4, leukotriene A 4, 5(S)-trans-5,6-oxido-7,9-trans11,14-c/s-eicosatetraenoic acid; L T B 4, leukotriene B4, 5(S),12(R)-dihydroxyo6,14-eis-8,!O-trans-eicosatetraenoic acid: SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Bistris. bis(2-hydroxyethyl)iminotris(hydro~methyl)methane: RP-HPLC, reverse-phase high-performance liquid chromatography; FPLC. fastprotein liquid chromatography; IPTG, isopropyl-fl-D-thiogalactopyranoside. Correspondence: A. Wenerholm, Department of Phys,t,~ogtc~l Chemistry, Karolinska lnstitutet, Box 60400, S. il~' 01 Stockholm,
Sweden.
human enzyme has been cloned from placenta and spleen c D N A libraries and sequenc~:d [4,5]. L T A 4 hydrolase is widely distributed, and is expressed also in cells apparently devoid of 5-1ipoxygenase/LTA 4 synthase activity [6-8]. Very little is known about the catalytic mechanism of LTA 4 hydrolase, but recently a sequence similarity between LTA 4 hydrolase and certain zinc metalloenzymes, for instance, aminopeptidase M and thelmolysin, was demonstrated, revealing the presence of a zinc binding motif in the enzyme [9,10]. Accordingly, human LTA 4 hydrolase was found to contain one atom of zinc per enzyme molecule [11,12]. In addition, the enzyme was recently shown to exhibit peptidase activity towards synthetic aromatic amides [12-14]. Mouse spleen LTA 4 hyd~ olase was recently cloned, sequenced and expressed in E. coli as an active fusion protein [15]. For studies on the effects of site directed mutagenesis on catalytic properties and metal content, we developed a rapid and reproducible purification procedure for recombinant mouse L T A 4 hydrolase. Physical and kinetic properties of the enzyme, including the zinc content and peptidase activity, are described.
97 Materials and Methods
LTA 4 methyl ester (Mcrck-Frosst, Canada) was saponified in tetrahydrofuran with 1 M LiOH (6% v/v) for 48 h at 4°C. ZnSO 4 and CoCI 2 (Johnson-Matthey specpurc) were dissolved in reagent grade Milli-Q water (Waters-Millipore). Alanine-4-nitroanilide, leucine4-nitroanilidc and i,i6-pheeanthroline were from Sigma. 4-Nitroaniline was from Kebo, Stockholm.
Enzyme purification Cell preparation and protein precipitations
Pharmacia). The column was equilibrated with 20 mM Tris-HCI (pH 8), containing 20% ammonium sulphate. Adsorbed proteins were eluted with a gradient from 20-0% ammonium sulphate. Active fractions were pooled and the buffer was changed to 25 mM Bistris (pH 7.1), by repeated centrifugation on a Ce,~tricon-30 microconcentrator (Amicon). For the final purification, a chromatofocusing column, Mono-P (5 x 200 mm, Pharmacia), was used. The starting buffer was 25 mM Bistris (pH 7.1), followed by 10% Polybuffer 74 (pH 4, pH adjusted with saturated iminodiacetic acid), at a flow rate of 0.5 ml/min. A peak exhibiting LTA 4 hydrolase activity was eh,ted at pH ( = p l ) 5.5. Th~ buffer was changed to 10 mM Tris-HCI (pH 8), by ultrafiltration as above, and aliquots of the purified enzyme were lyophilizcd and stored at -20°C. SDS-PAGE was performed on a Phast system (Pharmacia) using 10-15% gradient gels. The p l of the purified protein was determined by isoelectric focusing on a Phast IEF 4-6.5 gel. Bands of protein were visualized by staining with Coomassie brilliant blue. Protein concentrations were determined by the Bradlord method [16], using bovine serum albumin as standard. Zinc determinations were performed by atomic absorption spectrometry on a Perkin-Elmer Model 5000 electrothermal atomic absorption instrument. The result is an average of triplicate determinations on different dilutions of sample. Protein concentrations were determined by amino acid analysis.
Recombinant mouse LTA 4 hydrolase, a fusion protein with the first ten amino acids of the fl-galactosidase gene, was purified from E. coli (JM 101) transformed with the plasmid pULTA 4, the cDNA of which was cloned from a mouse splccn library [15]. Ceils were cultured at 37°C in M9 medium containing 0.2% casamino acids, 0.4% glucose, 2 mM MgSO4 and 75 # g / m l of ampicillin. IPTG (0.5 mM) was added at A~a~ ,,, ~ 0.2 and the incubation was allowed to continue for 2-3 h. Cells from 1 to 3 liter cultures were harvested by centrifugation (1000×g, 10 rain) and resuspended in 25 ml 50 mM Tris-HCl, (pH 8) supplemented with EDTA (5 mM), dithiothreitol (2 mM) phenyimethylsuifonyl fluoride (1 mM) and soybean trypsin inhibitor (60/.tg/ml), referred to as buffer A. All further mar~ipulations were carried out c,n ice or in a cold cabinet. Homogenization of cells was performed by sonication (4 x 15 s) using a Branson Sonifier, model S-125, at setting 4. The homogenate was centrifuged at 1 0 0 0 0 x g for 15 min and streptomycin sulphate (5% v / v of a 10% w / v solution) was added to the supernatant. Precipitated material was removed by centrifugation (10000 x g) and solid ammonium sulphate was added gradually to 40% saturation. Following another centrifugation, ammonium sulphate was added to 80% saturation and the resulting precipitate was collected by centrifugation. The 10000 X g pellet was resuspended in buffer A and subjected to ultrafiltration, using an Amicon PM-30 filter (Danvers MA), for buffer change (repeated three times).
Apo-LTA 4 hydrolase was prepared by treating the purified enzyme (0.5-1 mg, 1 mg / m l ) with 1,10phenanthroline (I0 mM) in 50 mM Hepes (pH 8), for 24-48 h at 4°C. The chelator was subsequently removed by ultrafiltration, as judged by the disappearance of absorbance at 264 nm [17]. The apoenzyme was stored in 10 mM Tris-HCl (pH 8) at 4°C and was stable for at least 2 weeks. For reconstitution with zinc or cobalt, the apoenzyme was treated with metal salt dissolved in 10 t~l Milli-Q water for 30 min at room temperature prior to determination of enzymatic activity.
Chromatographies, electrophoresis and zinc analysis
Determinations of enzyme activities
An anion exchange column (Mono-Q, 10 x 100 mm, Pharmacia) was equilibrated with 10 mM Tris-HCI (pH 8), at a flow rate of 3 m l / m i n and the protein obtained from precipitations was applied to the column. After elution of nonadsorbed material, a linear gradient of KCI was star~ed. Protein with LTA 4 hydrolase activity eluted between 0.10-0.15 M KCI. The Mono-Q pool was supplemented with 20% (w/v) ammonium sulphate and subjected to hydrophobic interaction chromatography on a Phenyl-Superose column, (5 x 50 mm,
The LTA 4 hydrolase activity was determined by incubating the enzyme (2-3 /~g in 100 #1 10 mM Tris-HCl, pH 8) with LTA 4 in 1 /.tl tetrahydrofuran (4-0 nmol). LTA 4 is a very labile comp~Jt,nd, which rapidly decomposes at high temperature and acidic pH. In order to reduce the influence of substrate instability in kinetic experiments, short-time incubations were performed on ice for 15 s. The reaction was quenched with 200 ttl of methanol and a defined amount of internal standard, prostaglandin B I (Upjohn), was
Preparation o1"apoenzyme
98 added. The samples were acidified to pH 3 with dilute HCI immediately bclorc RP-HPI,C on a column (Ultrosphere ODS. 250 × 4.6 ram) eluted with a mixture of a c e t o n i t r i l e / m e t h a n o l / w a t e r / a c e t i c acid (30: 35:35:0.01, v / v ) at a flow rate of 1 m l / m i n . The absorbance of the eluate (270 nm) was m o n i t o r e d continuously. Quantitations of LTB 4 were made by measurements of peak height ratios between LTB 4 and prostaglandin B 1, as described [18]. The peptidase activity was d e t e r m i n e d spectrophotometrically, essentially as described [19]. T h e substrates were dissolved in 50 m M Tris-HCI (pH 8) to a coucentration of 1 mM. Following addition of enzyme ( 1 - 4 #g). the product (4-nitroaniline) was m e a s u r e d as the increase in absorbanee at 410 nm, assuming an extinction coefficient of 8850 M - ~ c m - m. Alternatively, the assay was performed in the wells of a microtiterplate, and the absorbance at 405 nm was m e a s u r e d using a multiscan s p e c t r o p h o t o m e t e r , M C C / 3 4 0 (Labsystems). Quantitations were made from a standard curve obtained with known amounts of 4nitroaniline in 50 mM Tris-HCI (pH 8). Correction for spontaneous hydrolysis of the substrate was m a d e by subtracting the absorbance of control incubations without enzyme. Time-courses for the two enzymatic activities and effects of repetitive additions of LTA4 on the formation of LTB 4 and 4-nitroaniline were studied in the following way: LTA 4 hydrolase ( 2 5 / x g in 1 ml 10 mM Tris-HCI, p H 8) was kept on ice a n d an aliquot was removed for assay of peptidase activity, by incubation with alanine-4-nitroanilide. To the remaining solution, LTA 4 was added ( 8 0 / ~ M ) and aliquots were q u e n c h e d with methanol at the desired time points. After 5 min, an additional sample was removed for incubation with alanine-4-nitroanilide. A second dose of LTA,~ (80 # M , final concentration) was then a d d e d and the assays of LTB 4 formation and peptidase activity were repeated.
Mr (kD)
2
1
3
4
6
94.0 67,0
.....
43.0
30.0 20.1 14.4 Fig I. 5DS-PAGE of LTA 4 hydrolase at different steps of purificalion. Eleetrophoresis of the samples was carried out on a PhastGradient gel 10-15 (Pharmacia Phast System) and stained with Coomassie brilliant blue. The molecular mass markers were; phosphorylase b (94.(I kDa), bovine serum albumin (67.(I kDa), ovalbumin (43.0 kDa), carbonic anhydrase (30.0 kDak soybean trypsin inhibitor (20.1 kDa) and a-lactalbumin (14.4 kDa). The gel shows molecular weight markers (lane I), 100110xg supernatant (lane 21, 40-80r~ ammonium sulfate precipitate (lane 3), fractions from Mono-O (lane 4), PhenyI-Superose (lane 5) and Mono-P (hmc 6) chromatographies.
used as the final purification step (Fig. 3) a n d also by isoelectric focusing on a Phast I E F gel (data not shown). Four differen t enzyme batches were subjected to atomic absorption spectrometry which revealed the p r e s e n c e of 0.94, 0.66, 0.76 a n d 1.22 g-atom of zinc p e r mol of enzyme (Table 11). T h e m e a n value of these d e t e r m i n a t i o n s is 0.90 g - a t o m / m o l a n d since the theoretical value should bc an integer, tile closest approximation is one zinc atom p e r enzyme molecule.
(Nlla)2St)4 -20%
....
b
--....
Results
400-
/)l/ 3oot . l OOy
Purification and chemical properties of the enzyme R e c o m b i n a n t mouse L T A 4 hydrolase was purified to a p p a r e n t homogeneity, as judged by S D S - P A G E (Fig. 11. T h e procedure involved streptomycin a n d amm o n i u m sulphate precipitations, anion exchmJge, hydrophobic interaction (Fig. 2) and chromatofocusing chromatographies (Fig. 3) on FPLC. T h e enzyme was purified 19-fold with a yield of 60% (Table I). F r o m 1 I of cell culture, 1 - 2 mg of pure enzyme could be obtained. T h e molecular weight of the et,zyme was estimated to 69000 from S D S - P A G E (theoretical value 69860). The p l was d e t e r m i n e d to 5.5 by chromatofocusing,
5
- -
t
l0
I
l
20 Volume (ml) Fig. 2. PhenyI-Superose chromatography of mouse LTAa hydrolase. Enzymatically active fractions from Mono-O chromatography were pooled and 20% (w/v) solid ammonium sulfate was added. The column, PhenyI-Superose (5 x 50 mm), was equilibrated with 20 mM Tris-HCI, pH 8 with 20% (NH4)2 SO4 at a flow rate of 0.7 ml/min. After sample loading, a linear gradient of 20-0% ammonium sulfate was started. Aliquots (2 p,I) of the fractions were diluted to 100 p.I with 10 mM Tris-HCI, oH 8 and incubated with LTA 4 (35/zM) for 1 min at room temperature. The enzymatic product, LTB.;, was quantirated by RP-HPLC (see M~terials and Methods).
99 TABLE I Purification of recombinant mouse L 744 hvdrohtsc
LTA 4 hydrolase from 3 I cell culture was purified as dcscribed in Materials and Methods. A!iq'aols of the enzyme, afler indicated purification steps, were incubated with LTA 4 (45 /aM final c(mcenlration) for I rain at room Icmperature. Formation of [.TB 4 was analyzed by RP-IIPLC Fraction 1000(1× g-supernatan! Precipitations Mono-Q PhenyI-Superose Mono-P
Volume
Total protein
(ml)
(m~)
30
162 126 35 12.5 5.2
61)
5 5 2
Total activity (nmol/min/
I600 I 018 ~62
pH
7~\ .... IO(tO
--..
>
-5 750
g c
--
-4
{
Yield
Pu:ification
(r4)
(-fl)ld)
1011 87 78 64 60
I 3,5 S
10 II 3t~ 81
I 4110 I 256
Catalytic properties, thne-cour~es attd L T A 4 -mediated hlactit'ation A p p a r e n t v a l u e s o f K m a n d 1/",.... for t h e c p o x i d e h y d r o l a s e activity o f r e c o m b i n a n t m o u s e L T A 4 h y d r o lase w e r e d e t e r m i n e d f r o m i n c u b a t i o n s o f t h e e n z y m e (3 lag) with L T A 4 ( 5 - 1 0 0 taM'b for 15 s o n ice. T h e rate o f ~' l t..vl , , 4 p. .l g. u. u a' ~.t l.t.m. -__ at t h e d i f f e r e n t su', q r a t c c o n c e n t r a t i o n s w e r e e x t r a p o l a t e d to I m i n a n d t h e d a t a plott e d a c c o r d i n g to H a n e s . F r o m this plot K m a n d V, .... w e r e c a l c u l a t e d to 5 # M a n d 9(X) n m o l / m g p e r m i n , respectively (data not shown). T h e e n z y m e w a s a l s o f o u n d to exhibit p e p t i d a s e activity t o w a r d s s y n t h e t i c a m i d e s . T h e r e a c t i o n rate w a s c o n s t a n t for s e v e r a l h o u r s , w i t h o u t a n y sign o f i n a c t i v a t i o n (cf. Fig. 4B). T h e specific activity, u s i n g a l a n i n e - 4 - n i t r o a n i l i d c (i r a M ) as s u b s t r a t e , w a s calculated to 270 n m o l / m g p e r m i n at 37°C. A n o t h e r a m i n o acid c o n j u g a t e , l c u c i n c - 4 - n i t r o a n i l i d e , w a s also a s u b -
Specific actixit.~ (nmol/mg × rain)
185
;9
s t r a t e for m o u s e L T A 4 h y d r o l a s c , with a specific activity a p p r o x . 711% o f that o b t a i n e d with a l a n i n e - 4 - n i t r o anilide. A p p a r e n t K m a n d V,,~,X for t h e hydrolysis o f a l a n i n e - 4 - n i t r o a n i l i d e (at 37°C, p H 8) w e r e e s t i m a t e d f r o m H a n e s ' plot to 680 # M a n d 365 n m o l / m g p c min, r e s p e c t i v e l y ( d a t a not s h o w n ) . T h e i n c r e a s e in a b s o r b a n c e at 405 n m w a s r e c o r d e d d u r i n g a 30 m i n p e r i o d f r o m i n c u b a t i o n s o f e n z y m e (1 p.g) with s u b strate (0.06-1 mM). T h e t i m e - c o u r s e s a n d e f f e c t s o f repetitive a d d i t i o n s o f L T A 4 o n t h e two e n z y m a t i c activities a r e s h o w n in Fig. 4 A a n d B. T h e a m o u n t s o f L T B 4 i n c r e a s e d r a p i d l y a n d a l m o s t l i n e a r with t i m e d u r i n g t h e first 30 s a n d t h e n g r a d u a l l y levelled off. w h i c h in p a r t w o u l d be e x p e c t e d f r o m t h e instability o f t h e s u b s t r a t e w h i c h h a s a half-life at 4°C o f a b o u t 30 s [20]. F o l l o w i n g a s e c o n d d o s e o f L T A 4, o n l y s m a l l a d d i t i o n a l a m o u n t s o f L T B 4 w e r e p r o d u c e d (Fig. 4A). In this e x p e r i m e n t , also t h e p e p t i d a s e activity o f L T A 4 h.¢drolase w a s d e t e r m i n e d for aliqttots r e m o v e d b e f o r e a n d a f t e r a d d i t i o n s o f L T A 4 (Fig. 4B). F o r e a c h aliquot, t h e p e p t i d a s e activity w a s l i n e a r with t i m e for m o r e t h a n 2 h, w i t h o u t a n y sign o f i~metivation. H o w e v e r , in e n z y m e a l i q u o t s rem o v e d a f t e r a d d i t i o n s o f L T A 4, t h e p e p t i d a s e activities w e r e d e c r e a s e d ( a p p r o x . 5 0 % for e a c h d o s e o f L T A 4 ) , i n d i c a t i n g t h a t e x p o s u r e o f t h e e n z y m e to L T A 4 also a f f e c t e d s u b s e q u e n t p e p t i d a s e activity.
500
=
.
" ~
750
.,
500
~
E 0
0.0
I
I
I
I
0.5
1.0
1.5
2,0
Molar ratio metal/enzyme Fig. 5. Effects of zinc and cobalt on the peptidase activity of apo-LTA 4 hydrolase. APO-LTA4 hydrolase (3 /zg in 5 /~1 10 mM Tris-HCI, pH 8) was preincubated with increasing amounts of zinc (o) or cobalt (e) (10/J.I) in the wells of a microtiter plate After 30 rain, alanine-4-nitroanilide (1 mM in 50 mM Tris-HCI, pH 8. 235 #1) was added and the formation of 4-nitroaniline was recorded spectrophotometrically at 405 nm. Specific activities were calculated from 30 rain incubations at 22°C and represent means of duplicate samples.
E
N
¢ t~ +
0 r,..) +
0
Fig, ~. Specific epoxide hydrolase activities of apoenzyme, holoenzyme and apoenzyme reconstituted with zinc or cobalt. Apo-LTA 4 hydrolase (3 ~g)was preincubated with one equivalent of the desired metal in 100 ~l 10 mM Tris-HCI (pH 8) for 30 rain. Duplicate samples (3 /~g) of untreated apoenzyme, holoenzyme and reconstituted apoen~me were incubated with LTA 4 (80 p,M) for 15 s at 22°C for determination of the specific activities.
101
lasc. The enzyme was expressed irl E. coli and purified to apparent homogeneity by me;ms of precipitations and three different chromatograpifies on FPLC. with a yield of about 60%. The molecular weight was estimated to approx. 69000 and the p / w a s 5.5. With respect to the epoxide substrate LTA 4, apparent K m and V,.... were 5 u M and 900 n m o l / m g per min, (pH 8, 0°C), and thc time-course of the reaction was characterized by a rapid burst of catalysis, followed by reduced enzymatic activity upon a second dose of substrate. Thus, these physical and kinetic characteristics of recombinant mouse LTA4 hydrolase are similar to those reported for enzymes purified from other sources [3], with thc exception of guinea pig liver E T A 4 hydrolase, which exhibited a ~, .... significantly higher than the others
[21]. An additional enzymatic activity, i.e. hydroly;s of two synthetic amino acid dc,i~,ativcs (alaninc- and leucine-4-nitroanilide)was establ,shed t(~r mouse L] A4 hydrolase. Apparent K m and Vm,, (37°C, 50 mM TrisHCI, 13H 8) of the reactit~r, "~crc ~ctclmined to 680 ~M and 365 nmol/mg per rain, respectively, using alaninc4-nitroanilide as substrate. At a substrate concentration of 1 raM, the specific activity of this reaction was calculated to 270 nmol/mg per min, which can be compared to 380 nmol/mg per min obtained with human leukocyte LTA 4 hydrolase under the same conditions [13]. The recombinant mouse enzyme was shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The zinc atom was necessary for catalytic activity, since the apoenzyme was virtually devoid of both epoxide hydrolase and peptidase activity (Figs, 5 and 6), but could regain its enzymatic activities, towards both substrates, by addition of zinc or cobalt at a 1 : ! molar ratio. Therefore it seems likely that the two activities are exerted via closely related active sites. However, some differences between the two activities were also observed. Thus, enzyme containing cobalt exhibited a higher peptidase activity than the zinc enzyme (while the epoxide hydrolase activity was equal in both cases), and a 2-3 molar excess of zinc slightly inhibited the peptidase, but not the epoxide hydrolase activity. The putative zinc binding site in LTA 4 hydrolase has a very high similarity to the corresponding primary structure of certain other zinc hydrolases, for instance thermolysin, rat, E. coli and human aminopeptidase M, rat enkefalinase, P. aeruginosa elastase and human angiotensin converting enzyme [9,10, 22-26]. In a catalytic zinc site, the metal is coordinated in a tridentate complex to any three out of four amino acid residues from His, Cys, Glu and Asp. The first two ligands (L1 and L2), separated by a 'short spacer' of 1-3 amino acids, are believed to anchor the metal to the protein.
These primary ligands are separated from the third (L3) by a qong spaccr' of 21)-12(I amino acids [10. 27-29]. The fourth ligand is an activated t! !O molecule, which probably is involved in the catalytic reaction [29]. in accordance with these sequence homologies, the zinc atom in human, as well as mouse, LTAa hydrolasc would be coordinated to His-295, His-299 and Glu-318 [ I(1.11,15]. Interestingly, the zinc binding motif of LTA 4 hydrolase is located in the middle of a region of 108 amino acids which are I(X)% conserved between m o u ~ and man, implicating its importance for the enzyme function [15]. LTA 4 hydrolase has previously been shown to bc inactivated by its substrate, possibly by covalent binding of LTA 4 to the enzyme [3tL31]. Recently it was published that this suicide inactivation of LTA 4 hydrolase is intimately related to catalysis, and probably involves the active site [321. The peptidase activity was constant tot more than 2 h, without any sign of inactivation, while exposure of the enzyme to LTA 4, resulted in a dose-dependent decrease of both enzyme activities, although the epoxide hydrolase activity appeared to be more sensitive. Thus. the inhibitory effect of LTA4 on both enzyme activities also suggests that the corresponding catalytic sites are closely associated. The identification of LTA 4 hydrolase as a zinc metalloenzyme with peptidase activity will probably facilitate the development of drugs interfering with biosynthesis of LTB 4 and indeed, the peptidase inhibitor bestatin, and the angiotensin converting enzyme inhibitor captopril, were recently shown to inhibit both enzyme activities of LTA 4 hydrolase [14]. Other peptidases have been shown to take part in, for instance, degradation of peptides in the intestine [33]. processing of neurotransmittors [34], cleavage of angiotensin 11 to angiotensin II1 [35], and conversion of LTC 4 to LTE 4 [36]. The biological role of the peptidase activitx of LTA 4 hydrolase is presently unknown but the enzyme may have diverse functions, depending t~i its location and substrate availability.
Ackpowledgments The authors are greatly indebted to Ms. Eva Ohlson for excellent technical assistance. We also thank Dr. A.W. Ford-Hutchinson (Merck-Frosst, Canada) for the generous gift of ieukotriene A 4. This project was financially supported by the Swedish Medical Research Council (03X-217 and 03X-7467), Gustav V:s 80 ~rsfond, and O.E. and Edla Johanssons foundation.
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