In situ characterization of Helicobacter pylori arginase

Biochimica et Biophysica Acta 1388 (1998) 465^477

In situ characterization of Helicobacter pylori arginase George L. Mendz a

a;

*, Elizabeth M. Holmes a , Richard L. Ferrero

b

School of Biochemistry and Molecular Genetics, The University of New South Wales, Sydney, NSW 2052, Australia b Unite¨ de Pathoge¨nie Bacte¨rienne des Muqueuses (INSERM U389), Institut Pasteur, Paris 75724, France Received 8 June 1998; received in revised form 2 September 1998; accepted 9 September 1998

Abstract The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. One-dimensional 1 H- and 13 C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of L-arginine by the bacterium. Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates. A Km of 22 þ 3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains. Divalent cations stimulated arginase activity, and the most potent activators were Co2‡ s Ni2‡ s Mn2‡ . The activity was highly specific for L-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin. The Ki of several inhibitors was measured and served also to characterize the enzyme activity. The presence of bicarbonate enhanced the hydrolysis of L-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations. Amino acid sequence analyses revealed important differences between the deduced structures of H. pylori arginase and those of other organisms. This finding was consistent with experimental data which showed that H. pylori arginase has unique properties. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Arginase; Microaerophily; NMR spectroscopy; (Helicobacter pylori)

1. Introduction Arginases (EC 3.5.3.1) are urea-cycle enzymes catalyzing the hydrolysis of arginine to ornithine and urea, and are found in organisms as diverse as bacteria and vertebrates. These enzymes play a fundamental role in nitrogen metabolism by splitting urea from arginine, its carrier molecule, and thus producing a small, neutral, highly soluble and nitrogen rich molecule easily excreted by organisms.

* Corresponding author. Fax: +61 (2) 9385-1483; E-mail: [email protected]

The wide distribution of the arginase protein family in prokaryotes and eukaryotes is re£ected by its close association with biological evolution. Owing to the ubiquity of these enzymes it has been proposed that a member of this family was part of the genome of the universal common ancestor [1]. Arginases from di¡erent sources vary in their amino acid sequences and properties [2,3]. Comparative studies of arginases are beginning to elucidate the speci¢c physiological roles of these enzymes in di¡erent organisms [4]. Helicobacter pylori is a Gram-negative, microaerophilic, vibrioid bacterium which occupies a unique ecological niche in the upper gastrointestinal tract

0167-4838 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 8 3 8 ( 9 8 ) 0 0 2 0 7 - 6

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[5]. The bacterium has been established as the major etiological agent of acute chronic gastritis, associated with peptic ulcers, and more recently linked to the development of gastric cancer [6]. In its natural habitat on the lining of the stomach gastric epithelium, H. pylori is exposed to relatively high concentrations of host breakdown products including amino acids [7], and laboratory-adapted and low-passage H. pylori strains grow and proliferate in media containing amino acids as sole basic nutrients, with L-arginine and six other amino acids being an essential requirement for growth [8,9]. To understand the physiology of the bacterium it is necessary to characterize the metabolic processes involved in the utilization of amino acids. Several amino acids including L-asparagine, L-aspartate, and L-serine are utilized at fast rates in liquid cultures, with production of considerable quantities of free ammonium [10]. L-Arginine is catabolized rapidly to L-ornithine and urea in cell suspensions or lysates of H. pylori, and the utilization of L-arginine takes place via the arginase pathway [11]. It has been established also that the bacterium has a complete urea cycle [12]. The initial utilization of L-arginine by H. pylori was investigated in situ by measuring the characteristics of arginase activity in metabolically competent cells and lysates, with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium. Nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein puri¢cation techniques were employed to study, amongst other properties, the cellular location, kinetic parameters, divalent cation requirements, speci¢city and e¡ects of inhibitors and bicarbonate on arginase activity. In situ studies of enzyme activities are required to obtain a cogent view of the physiology of microorganisms. The activities of enzymes in the intracellular milieu depend on the intrinsic physicochemical properties of these proteins and on their interactions with other cellular components. Modulation of enzyme activities of functional relevance occur not only amongst the constituents of recognized multi-enzyme clusters, but also amongst soluble enzymes that can be extracted from ruptured cells. Thus, in situ investigations of enzyme activities may reveal properties which otherwise would remain undetected in studies

with puri¢ed proteins. For these reasons, the initial utilization of L-arginine by H. pylori has been investigated in cells and lysates. 2. Materials and methods 2.1. Materials Blood Agar Base No. 2, Columbia Agar Base, Isosensitest Broth and horse serum were from Oxoid (Basingstoke, UK). Bovine serum albumin Fraction V, bovine liver catalase, NK-acetyl-L-arginine, NKcarbamoyl-L-arginine, L-argininamide, D-arginine, Larginine, L-argininic acid, L-arginine hydroxamate, Larginine ethyl ester, L-arginine methyl ester, L-arginine phosphate, L-canavanine, guanidino butyrate, homoarginine, Ng -nitro-L-arginine, putrescine, were from Sigma (St. Louis, MO); L-[U-14 C]arginine (270 mCi/mmol) from ICN Biomedicals (Seven Hills, NSW, Australia); amphotericin B (Fungizone) from Squibb (Princeton, NJ); vancomycin from Eli Lilly (West Ryde, NSW, Australia); Sterile 0.2 Wm Minisart ¢lters were from Sartorius (Go«ttingen, Germany), and self-vented tissue culture £asks and sterile microtiter trays from Corning (Corning, NY). All other reagents were of analytical grade. 2.2. Bacterial cultures and preparation H. pylori laboratory-adapted strains NCTC 11639, N6, N6ureG: :Km and UNSW P10, as well as lowpassage wild-type strains UNSW 10536 and UNSW 10593/5 were grown on Blood Agar Base No. 2 plates supplemented with 5% (v/v) horse blood, polymyxin B (1.25 U/l), trimethoprim (5 mg/l), vancomycin (10 mg/l) and amphotericin B (2.5 mg/l). Cultures were passaged every 24^30 h and incubated in a Forma Stericult incubator (Marietta, OH) in an atmosphere of 10% CO2 in air, 95% humidity at 37³C. Cells were harvested in log phase (ca. 24 h) in sterile NaCl (150 mM), checked for purity under phase contrast microscopy, and tested for urease and catalase activities. Cells were washed three times by centrifuging at 17 000Ug for 3 min at 6³C. The supernatants were collected and the pellets resuspended in saline. After the ¢nal wash, packed cells were resuspended in saline and employed as inoculum for dif-

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ferent experiments. Cell lysates were prepared by resuspending packed cells in sterile NaCl (150 mM) or KCl (150 mM) containing 109 ^1010 cells/ml and lyzed by two freeze^thaw cycles in liquid nitrogen. To separate supernatant and particulate fractions, bacterial suspensions were centrifuged at 20 000Ug for 10 min at 6³C. 2.3. NMR spectroscopy Metabolically competent H. pylori cells and lysates generate ornithine and urea from arginine as the sole substrate indicating the presence of arginase activity in the bacterium [12]. Samples that had been prepared in either NaCl (150 mM; packed cells, lysates or membrane fractions) or in KCl (150 mM; lysates or membrane fractions), were placed in 5 or 10 mm tubes (Wilmad, Buena, NJ). The substrate, at concentrations between 1 and 100 mM, was added to start the reactions. Measurements were carried out at 37³C. 1 H-NMR free induction decays were collected using a Bruker DMX-500 or a Bruker DMX-600 spectrometer, operating in the pulsed Fourier transform mode with quadrature detection. Proton spectra were acquired with presaturation of the water resonance. The instrumental parameters for the DMX-500 instrument were: operating frequency, 500.13 MHz; spectral width, 5000 Hz; memory size, 16K; acquisition time, 1.638 s; number of transients, 48^144; pulse angle, 50³ (3 Ws); and relaxation delay with solvent presaturation, 1.0 s. The instrumental parameters for the DMX-600 instrument were: operating frequency, 600.13 MHz; spectral width, 6009.61 Hz; memory size, 16K; acquisition time, 1.363 s; number of transients, 48^144; pulse angle, 50³ (3 Ws); and relaxation delay with solvent presaturation, 1.6 s. Spectral resolution was enhanced by Gaussian multiplication with line broadening of 30.7 Hz and Gaussian broadening factor of 0.19. Chemical shifts are quoted relative to sodium 4,4-dimethyl-4-silapentane-1-sulfonate at 0 ppm. One-dimensional natural abundance 13 C-NMR spectra were acquired with composite pulse decoupling using a Bruker ACP-300 spectrometer. The instrumental parameters were: operating frequency, 75.5 MHz; spectral width, 16129 Hz; memory size, 16K; acquisition time, 1.508 s; number of transients, 2000; and pulse angle, 66³ (9 Ws). Ex-

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ponential ¢ltering of 3 Hz was applied prior to Fourier transformation. 13 C Chemical shifts are quoted relative to L-Arg-K-CH at 55.32 ppm. The time-evolution of substrates and products were followed by acquiring sequential spectra of the reactions. Progress curves were obtained by measuring the integrals of substrate and product resonances at each point in time. Maximal rates were calculated from good ¢ts (correlation coe¤cientsv0.99) of the data to straight lines for 30^80 min of the incubations. Calibrations of the peaks arising from substrates were performed by extrapolating the resonance intensity data to zero time and assigning to this intensity the appropriate concentration value. The intensity of resonances corresponding to products were calibrated by adding the appropriate metabolite to cell suspensions and constructing standard concentration curves. 2.4. Partial puri¢cation of H. pylori arginase and colorimetric assay of enzyme activity Partial puri¢cation of arginase was performed in lysates obtained by suspending bacterial cells in TrisHCl (20 mM, pH 7.5), L-arginine (20 mM), EDTA (1 mM), and L-mercaptoethanol (1 mM) bu¡er and sonicating with a microtip in a Branson Soni¢er 450 (Danbury, CT) for 1 min/ml of suspension at a power level of 4^5 and 40% duty cycle. Lysates were centrifuged at 2³C and 27 290Ug for 20 min in a Hereus Sepatech centrifuge. The supernatant was collected, streptomycin sulfate (0.15 g/mg protein) was added and the mixture stirred gently at 4³C for 30 min. The resulting suspension was centrifuged as before, the supernatant retained, and protein precipitation carried out at di¡erent ammonium sulfate concentrations; arginase activity was found in the 20^50% fraction. Enzyme activity was assayed by a method adapted from that of Mia and Koger [13]. The ammonium sulfate fraction containing arginase activity was desalted using a Pharmacia PD10 column (Boronia, VIC, Australia) with Tris (100 mM, pH 7.4) bu¡er. The protein concentration was 1.5 þ 0.1 mg/ml. CoCl2 (5 mM) was added to the protein extract and the mixture incubated at 55³C for 5 min. Arginase extract (20 Wl) was added to 0.5 ml pre-warmed Tris (100 mM, pH 7.4) bu¡er containing 5 mM

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L-arginine

and incubated for 1 h at 37³C. To stop the reaction 2 ml of ninhydrin reagent (7.5 g/l ninhydrin HCl in a 91:7.9:1.1 (v/v/v) mixture of glacial acetic acid, distilled water and of 85% phosphoric acid) were added, color was developed for 20 min at 95³C, and was stable for about 30 min after that. Samples were cooled prior to measuring their absorbance at 515 nm. The reference sample was prepared by omitting the incubation at 37³C. 2.5. Kinetic parameters of arginase Michaelis constants (Km ) and maximal velocities (Vmax ) for arginine conversion to ornithine by suspensions of bacterial cells or lysates were determined from the maximal rates of arginase activity of ten time courses. Rates were measured employing 1 H-NMR spectroscopy by measuring the decrease of arginine levels. The values of the kinetic parameters and standard errors were calculated by non-linear regression analysis employing the program Enzyme Kinetics (Trinity Software, Campton, NH, USA).

2.7. E¡ects of divalent cations and speci¢city of arginase activity The e¡ects of nine divalent cations on enzyme activity and partially puri¢ed arginase were studied by adding to the assay mixtures the chloride salts of the cations at concentrations up to 10 mM. The speci¢city of the arginase activity was investigated in bacterial lysates by measuring the catabolism of 16 compounds with similar structures to L-arginine, and by competition experiments between these compounds and the L-amino acid. The metabolites employed included the D-enantiomer, and compounds with substituents in the amino, carboxylic or guanidino end groups, and modi¢cations of the aliphatic chain. 2.8. E¡ects of inhibitors on enzyme activity The e¡ects of potential inhibitors on arginase activity were investigated by measuring the rates of Larginine conversion to ornithine in suspensions of bacterial lysates employing 1 H-NMR spectroscopy. At substrate concentrations below the Km the inhibition constant can be calculated from the expression vo =v ˆ 1 ‡ I=K i ;

2.6. Molecular size of the catalytic complex An approximate size of the catalytically active complexes was investigated by membrane ¢ltration. Desalted protein extract (1 ml) was suspended in EDTA (1 mM) and ¢ltered through a 10 kDa cuto¡ membrane (Amicon, YM10) using an ultra¢ltration cell (Amicon, Beverly, MA); retentate and ultra¢ltrate fractions were collected. The retentate was washed three times by the same procedure and resuspended in 1.5 ml Tris (100 mM, pH 7.4) bu¡er. The ¢ltrate was concentrated through a 1 kDa membrane (Amicon, YM1), and resuspended in 1.5 ml Tris (100 mM, pH 7.4) bu¡er. Each fraction was tested for arginase activity employing the colorimetric method. Several fractions were obtained from the 10 kDa membrane retentate by ¢ltration through 30, 50, 100 and 300 kDa cut-o¡ Amicon membranes. Retentates and ¢ltrates were collected after each ¢ltration, the volumes of each suspension restored to the original starting value following the procedure indicated above, and each fraction tested for arginase activity by the colorimetric method.

where vo and v are the uninhibited and inhibited rates of hydrolysis, respectively, and I is the concentration of inhibitor [14]. 2.9. L-Arginine transport in the presence of ornithine or sodium bicarbonate Transport of L-arginine into H. pylori NCTC 11639 cells was measured at 24³C employing L-[U14 C]arginine and the centrifugation through oil method previously described [15]. Transport of 5 mM L-arginine into cells suspended in phosphate bu¡ered saline (pH 7.4) was linear for 120 s. In£ux rates of 1 mM L-arginine were determined at a ¢xed timepoint of 20 s in the presence of ornithine at concentrations between 0 and 20 mM or sodium bicarbonate at concentrations between 0 and 30 mM. Measurements were performed in two ways, either by placing ornithine or bicarbonate in the permeant containing L-arginine and then mixing with the cells, or by mixing the cells with the permeant and immediately adding ornithine or sodium bicarbonate.

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2.10. Analyses of the arginase sequence Computer analyses of the arginase amino acid sequence deduced from the H. pylori genome database [16] were carried out with programs of the National Center for Biotechnology Information (Paris, France). Comparison of H. pylori arginase with sequences in the data bank was carried out with the BLASTP alignment tool enhanced with the BEAUTY tool on the BCM Search Launcher server [17]. Sequence similarities were determined also with the PILEUP tool from the Wisconsin sequence analysis package (Genetics Computer Group). Molecular weights and isoelectric points were determined using the EXPASY tool on the Molecular Biology Server. A survey of amino acid motifs was performed using the MOTIFFINDER tool. Protein homologies were calculated with BESTFIT from GCG. 2.11. Protein determination Estimation of the protein content of samples was made by the bicinchoninic acid method employing a microtiter protocol (Pierce, Rockford, ILL, USA). 3. Results The metabolism of arginine by H. pylori was followed in time courses employing 1 H- or 13 C-NMR spectroscopy, and the rates of L-arginine conversion to L-ornithine and urea were measured in suspensions of bacterial cells or lysates in isotonic NaCl or KCl. The assignment of resonances in these metabolic reactions has been described previously [11]. In incubations of L-arginine with H. pylori cells or lysates, the 1 H-NMR resonances arising from the aliphatic K, L, Q and N protons changed with time in the sequential spectra of the suspensions (Fig. 1). Owing to the overlap between the K, L, and Q resonances of the arginine substrate and the ornithine product, rates of arginine hydrolysis were measured by the decline of the intensity of the resonance corresponding to the N-proton. The six 13 C-NMR resonances arising from the carboxylic, guanidino j, and aliphatic K, L, Q and N carbon atoms of arginine decreased with time in the sequential spectra of incubations with H. pylori suspensions, and ¢ve other

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resonances at 174.97, 55.20, 40.01, 28.40, and 23.74 corresponding to L-ornithine emerged (Fig. 2). The best data for determining arginine hydrolysis rates with 13 C-NMR spectroscopy were obtained by measuring the decline of the N-carbon resonance. Arginase activity was associated with the particulate fraction obtained by centrifugation of bacterial cells disrupted by freeze^thawing. This is a gentle procedure for rupturing cells which appears to open holes in the cell envelope and have minimal e¡ects on its overall integrity. Enzyme activity was observed in the supernatant fraction of cells broken by sonication, a more vigorous method which produces cell debris of di¡erent sizes, including vesicles. In competition experiments, the rate of L-arginine transport into H. pylori cells was not a¡ected by the presence of a 20-fold concentration of ornithine, indicating that the hydrolysis of arginine took place inside the cells. 3.1. Kinetic parameters of arginase activity Similar Km and Vmax for arginase were determined in cell or lysate suspensions from measurements of maximal rates of L-arginine utilization in time courses studies at substrate concentrations between 2 and 80 mM (Table 1). No signi¢cant di¡erences were found between the Km of the strains tested. The low-passage strain showed a somewhat higher maximal velocity than the laboratory strains (Table 1). 3.2. E¡ects of divalent cations and size of the catalytic complex A common feature of most arginases, whether of prokaryotic or eukaryotic origin, is a requirement for divalent cation activators. Arginase activity was observed in H. pylori cell suspensions or lysates prepared by freeze^thawing in the absence of additional divalent cations in the medium. In lysate suspensions, the presence of cations a¡ected enzyme activity, whilst the addition of Co2‡ , Mg2‡ , Mn2‡ or Ni2‡ , each at 1 mM concentration, to cell suspensions did not a¡ect the rates of arginase activity in these samples. Measurement by 1 H-NMR spectroscopy of the e¡ects of nine divalent cations on arginase activity in H. pylori lysates indicated that the

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Fig. 1. 1 H-NMR spectra of a time course of the conversion of L-arginine by H. pylori NCTC 11639 cells suspended in NaCl (150 mM). The substrate concentration was 10 mM. The resonances corresponding to the K, L, Q and N protons of L-arginine are indicated on the bottom spectrum, the resonance corresponding to the N protons of L-ornithine is indicated on the top spectrum. The dashed line indicates the disappearance of the Arg-N resonance, and the solid line the appearance of the Orn-N resonance. The time at which each spectrum was acquired is shown on the right-hand side.

Fig. 2. 13 C-NMR spectra of a time course of the conversion of L-arginine by H. pylori NCTC 11639 pellet fraction suspended in NaCl (150 mM). The substrate concentration was 40 mM. The resonances corresponding to the K, L, Q and N carbons of L-arginine are indicated on the bottom spectrum, the resonance corresponding to the N carbon of L-ornithine is indicated on the top spectrum. The time at which each spectrum was acquired is shown on the right-hand side.

most potent stimulators were Co2‡ s Ni2‡ s Mn2‡ (Table 2), whereas Cd2‡ , Cu2‡ and Zn2‡ inhibited the enzyme activity. In lysates suspended in NaCl (150 mM) and the metal chelator EDTA (0.25 mM), arginase activity at 100 mM arginine concentration decreased to 11% of the value in the absence of the chelator. Addition of CoCl2 (0.5 mM) reversed the inhibition of EDTA increasing the enzyme activity to 68% of the original

value. The results suggested that Co2‡ ions substituted the chelated metal ions in the arginine binding site. The e¡ects of divalent cations were measured also in partially puri¢ed arginase preparations suspended in Tris bu¡er at pH 7.4 employing the colorimetric method. The order of activation was also Co2‡ s Ni2‡ s Mn2‡ in these enzyme preparations. The results of membrane ¢ltration experiments in-

Table 1 Kinetic parameters for arginase activities in cell and lysate suspensions in NaCl (150 mM) of four H. pylori strains Strain

Cells

Lysates

Km (mM)

Vmax (nmol/min/mg protein)

Km (mM)

Vmax (nmol/min/mg protein)

NCTC 11639 N6 N6ureG: :Km UNSW 10593/5

24 þ 2 21 þ 2 20 þ 2 25 þ 3

152 þ 14 102 þ 10 105 þ 10 193 þ 18

23 þ 2 20 þ 2 21 þ 2 22 þ 2

155 þ 14 108 þ 10 109 þ 10 200 þ 19

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G.L. Mendz et al. / Biochimica et Biophysica Acta 1388 (1998) 465^477 Table 2 Arginase activities of H. pylori strain NCTC 11639 lysates suspended in 150 mM NaCl, 30 mM Tris-HCl bu¡er and 100 mM L-arginine in the presence of 1 mM of the chloride salt of divalent cations (n = 3) Cation

Activity (%)

None Cadmium Calcium Cobalt Copper Iron Magnesium Manganese Nickel Zinc

100 0 190 1200 0 175 140 360 500 0

Errors were estimated at þ 15%.

dicated the existence of inactive arginase subunits with a size 30^50 kDa that could be converted to the catalytic form of the enzyme by incubation with divalent cations. The size of the active complex was determined to be between 100 and 300 kDa. 3.3. Speci¢city of arginase activity Arginase activity in H. pylori lysates was not observed with D-arginine. Other compounds used to

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investigate the speci¢city of the arginase were metabolites with changes in the K-amino group including NK -acetyl-L-arginine, NK -carbamoyl-L-arginine and L-argininic acid; metabolites with modi¢cations in the carboxylic acid group were agmatine, L-argininamide, L-arginine ethyl ester, L-arginine hydroxamate, and L-arginine methyl ester; and compounds with substitutions in the guanidino group were L-arginine phosphate, L-citrulline and Ng -nitro-L-arginine. The metabolites with modi¢cations in the aliphatic chain were L-canavanine, guanidino butyric acid and L-homoarginine. Hydrolysis of the ester bonds of L-arginine methyl ester or L-arginine ethyl ester was observed in bacterial lysates. 31 P-NMR spectroscopy data indicated that the high energy bond of L-arginine phosphate was hydrolyzed in the presence of lysates yielding inorganic phosphate. None of the metabolite analogs, with the exception of L-argininamide, was catabolized by lysate suspensions under the same conditions as those employed to measure arginase activity. To ascertain whether compounds a¡ected the rate of arginase activity, competition experiments were performed in lysate suspensions incubating L-arginine together with another metabolite analog, each at 20 mM concentration. The results in Table 3 indicated that most of the analogs did not a¡ect argi-

Table 3 Arginase speci¢city and activity of H. pylori strain NCTC 11639 lysates suspended in 150 mM NaCl, 0.1 mM CoCl2 . The speci¢city was determined in suspensions each containing 20 mM of the metabolites Metabolite

Substrate activity

Activity (%)

L-Arginine

Yes No No No Yes No No No No No No No No No No No

100 100 104 103 100 99 56 96 97 109 42 100 100 100 85 100

D-Arginine

NK-Acetyl-L-arginine Agmatine L-Argininamide L-Arginine ethyl ester L-Arginine hydroxamate L-Arginine methyl ester L-Arginine phosphate L-Argininic acid L-Canavanine NK-Carbamoyl-L-arginine L-Citrulline Guanidino butyric acid L-Homoarginine Ng -Nitro-L-arginine

The activity of the enzyme was measured in competition experiments in the presence of 20 mM L-arginine and 20 mM of each analog (n = 3). Errors were estimated at þ 15%.

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Table 4 Inhibition constants of several inhibitors of arginase activity in H. pylori strains NCTC 11639 and N6ureG: :Km (*) lysates suspended in 150 mM NaCl with 5 mM L-arginine Inhibitor

Ki (mM)

Acetyl hydroxamate Ammonium EDTA L-Ornithine Phosphate Urea*

6.2 þ 0.4 35 þ 6 0.29 þ 0.03 No inhibition 135 þ 15 66 þ 7

nase activity with the exception of L-arginine hydroxamate, L-canavanine and L-homoarginine. 3.4. E¡ects of inhibitors The e¡ects of eight potential inhibitors of arginase activity were investigated in suspensions of lysates in NaCl. p-Chlorobenzoate and iodoacetamide inhibited the activity of the enzyme in situ at micromolar concentrations. The inhibition constants of the other six compounds are given in Table 4. In lysates of strain NCTC 11639 the presence of urea produced biphasic concentration-dependent e¡ects on arginase activity. Urea added at low concentrations enhanced arginase activity; the activation was approximately 80% at 10 mM urea. Increasing the concentration of exogenous urea above 10 mM resulted in a reduction in arginase activity from the maximum activation. Given that H. pylori strains hydrolyze urea and

Fig. 3. Rates of L-arginine hydrolysis by H. pylori NCTC 11639 cells as a function of added sodium bicarbonate. Bacteria were suspended in NaCl (150 mM) and the initial L-arginine concentration was 10 mM.

yield ammonium and bicarbonate as products, Ki determinations for urea were performed using lysates of the urease-negative N6ureG: :Km H. pylori mutant (Table 4). The inhibition constants spanned several orders of magnitude for the compounds tested. 3.5. In situ modulation of arginase activity by bicarbonate Rates of arginase activity in H. pylori cells suspended in NaCl (150 mM) increased linearly with NaHCO3 concentration up to 25 mM (Fig. 3). At 10 mM L-arginine and 30 mM bicarbonate, the increases measured for strains NCTC 11639, N6,

Table 5 Deduced molecular weight, percent similarity and identity, and pI of arginase amino acid sequences with the closest homologies to H. pylori, found employing the BLASTP and BEAUTY search and alignment tools Organism

MW

Identity (%)

Similarity (%)

pI

H. pylori (AEOOO639) B.subtilis (X81802) B. caldovelox (U48226) S. pombe (X75559) R. norvegicus (I, A26702) R. norvegicus (II, U90887) B. abortus (U57319) H. sapiens (I, X12662) H. sapiens (II, D86724)

36 925 32 154 32 433 35 721 34 999 38 640 33 182 34 734 38 578

100 30.3 26.6 26.0 25.2 24.7 22.9 22.8 21.0

100 43.2 40.8 34.9 36.1 34.2 35.7 34.6 32.1

6.34 5.10 5.58 5.55 6.76 6.24 5.70 7.09 6.00

GenBank accession numbers are given in brackets next to the name of the organism. Molecular weights (MW) and isoelectric points (pI) were determined using the EXPASY tool.

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N6ureG: :Km, UNSW P10 and UNSW 10536 were 45, 50, 50, 30 and 150 þ 10%, respectively. In lysates suspended in NaCl and 10 mM L-arginine, addition of NaHCO3 decreased enzyme activity. In the presence of 30 mM bicarbonate, arginase activity in strain NCTC 11639 lysates were reduced to 78 þ 10% of the controls without NaHCO3 . The activity measured in the presence of 30 mM bicarbonate and 0.1 mM CoCl2 was 92 þ 10% of the controls without bicarbonate. Similarly, the activity of partially puri¢ed arginase measured by the colorimetric method in the presence of 5 or 10 mM CoCl2 , was not a¡ected by the addition of NaHCO3 at concentrations up to 30 mM. These results indicated that the catalytic form of H. pylori arginase did not appear to be a¡ected by NaHCO3 . The metal ions in arginases contribute to the assembly of the catalytic complex. Since the inhibitory e¡ect observed in lysates in the absence of cobalt was partially reversed by addition of metal ions, it is reasonable to suggest that one of the e¡ects of bicarbonate anion may be hindering the formation of the active complex. The in£ux of arginine into H. pylori cells was measured to ascertain whether the e¡ect of bicarbonate on arginase activity in intact bacterial cells was related to the transport of arginine across the membrane. The rate of uptake of 1 mM L-arginine into H. pylori NCTC 11639 cells at 24³C was linear with permeant concentration, and was not a¡ected by the presence of NaHCO3 up to 30 mM concentration. The data indicated that bicarbonate did not a¡ect the transport of L-arginine. 3.6. Arginase activity and cell age The activity of arginase decreased with the age of the cells. In the presence of 120 mM L-arginine and 1 mM CoCl2 , the enzyme activities of lysates, prepared from cells grown for 24, 48 and 72 h, were measured by 13 C-NMR spectroscopy and found to be of the order of 168 þ 21, 153 þ 18 and 87 þ 10 nmol/min/mg, respectively (n = 2). A stronger decline of arginase activity with cell age was observed in the absence of the cobalt salt; at 24, 48 and 72 h the rates were 12.4 þ 1.6; 6.6 þ 0.8 and 3.9 þ 0.5 nmol/min/ mg, respectively (n = 2). The morphology of bacteria in the cultures was examined by phase-contrast microscopy. Cells harvested after 24 h incubation had a

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rod-shaped bacillary form; after 48 h incubation, between 40 and 60% of the cells were in the coccoid form; and after 72 h incubation more than 95% of the cells were coccoid. 3.7. Comparison of H. pylori arginase amino acid sequence with other enzyme sequences The deduced amino acid residue sequence of H. pylori arginase [16] was compared to the sequences in the data banks employing the BLASTP, BEAUTY and PILEUP search and alignment tools. The degrees of identity and similarity between the arginase of H. pylori and those of other organisms was relatively low, with the arginases of Bacillus subtilis, Bacillus caldovelox, Schizosaccharomyces pombe, Bacillus abortus and rat and human type I and type II, being the most similar to the enzyme of H. pylori. There are at least two isoforms of mammalian arginases classi¢ed according to their properties as type I (hepatic) and type II (non-hepatic), and coded by two di¡erent genes [3,18]. The deduced molecular weights, isoelectric points, and the percent identity and similarity between the H. pylori and each of the other enzymes are given in Table 5. A comparison of 15 arginase sequences comprising the ones analyzed in this study, excluding the H. pylori and B. abortus sequences, identi¢ed 25 residues conserved in all the enzymes [3]. Included amongst these were three histidines and four aspartates shown to be important for catalysis and stability of the binuclear metal center [19,20]. The 25 residues were found to be conserved also in the B. abortus sequence. In the H. pylori sequence, the seven important residues His91, -118 and -133, and Asp-116, -120, -234 and -236 were conserved; but only 11 of the other 18 residues were identical. The segment ILYLDAHAD comprising residues 112^120 of the H. pylori sequence showed the signature pattern of type II arginases, a motif of conserved regions that contain charged residues potentially involved in binding and/or catalysis [1]. The region 153^165 appeared as an insertion in the H. pylori sequence and was not found in the alignments with the other sequences. Secondary structure predictions of the deduced amino acid sequence with the algorithms SIMPA96 and PREDATOR indicated a helical conformation for the ¢rst 11 residues of this segment [16]. X-ray di¡raction

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analysis revealed 10 K-helices in the topology of rat liver arginase [20]. These helices can be located in the predicted secondary structure results of the H. pylori sequence, with the region 153^163 constituting an additional helix not found in the rat hepatic arginase. 4. Discussion Arginase activity has been characterized in several bacterial genera including bacilli, cyanobacteria and mycobacteria. In some cases, expression of the enzyme is induced by L-arginine and other polyamines, and arginase activity is often subject to carbon or nitrogen catabolite control [21^24]. L-Arginine is an essential amino acid for growth of H. pylori in vitro [8,9], and its catabolism is initiated by arginase, suggesting that the enzyme activity is constitutive in this bacterium. This conclusion is supported by the observation that the enzyme activity was present in all laboratory-adapted and low-passage strains investigated in this and previous studies [11,12]. Ureotelic organisms, such as mammals, excrete most of their nitrogen as urea, and uricotelic animals such as birds and some reptiles excrete nitrogen primarily as uric acid. Arginases are designated also as ureotelic or uricotelic on the basis of their quaternary structure and kinetic parameters. Ureotelic arginases are complexes of lower molecular masses (120^150 kDa) and Michaelis constants (1^25 mM) than those of the uricotelic type, which have sizes of 200^270 kDa and Km of 100^200 mM [4]. Separation of partially puri¢ed H. pylori enzyme preparations into fractions indicated the presence of subunits with a size 30^50 kDa, which is in good agreement with the 36.9 kDa molecular mass estimated from the deduced amino acid sequence [16]. The size of the H. pylori arginase active complex was between 100 and 300 kDa, but this information is insu¤cient to classify the enzyme. The subunit size of H. pylori arginase compared well with those of other ureotelic microorganisms; it was larger than the arginases of B. abortus, B. subtilis [25], B. caldovelox [26] and Rhodobacter capsulatus E1F1 [24], similar to those of Agrobacterium tumefaciens [27], and S. pombe [28], and smaller than the enzymes of Bacillus anthracis [29] and Saccharomyces cerevisiae [30]. The subunit

size of H. pylori arginase was larger than the mammalian hepatic type arginases and slightly smaller than those of the non-hepatic enzymes from human, murine and rat sources [31^33]. The Km of H. pylori arginase activity suggested that it was of the ureotelic type; it was an order of magnitude larger than those determined for other bacilli [26,29], and mammals [34], similar to those of R. capsulatus E1F1 [24], and S. cerevisiae [35], and much lower than the arginases of Neurospora crassa [36], Pista paci¢ca [37] and chicken liver [38]. The H. pylori enzyme activity was associated with the cell envelope fraction obtained by centrifugation of lyzed cells, and the data on arginine transport in competition with excess ornithine suggested an inner membrane surface location for arginase. Mammalian type I arginases appear to be cytosolic, although there have been di¡erent claims about the exact locations of these enzymes [3,4]. Numerous studies have indicated that type II arginases may be located in the mitochondrial matrix [39]. Comparison of human hepatic-type and non-hepatic type arginases indicated that the type II arginases are 32 residues longer than type I owing to the presence of additional residues at both termini. It has been suggested that the additional N-terminal residues, which do not include acidic residues, are probably involved in the mitochondrial localization of the enzyme [3]. The size of H. pylori arginase is closer to those of type II arginases, but it contains several acidic residues near its N-terminus. On the other hand, secondary structure predictions suggest a helical region in residues 153^163, within the segment 153^165 found in the H. pylori protein and not present in the other known sequences. A possible role of this putative helix may be to interact with the membrane, thus resulting in the association of the enzyme with the cell envelope. H. pylori arginase appeared to be a metalloprotein, but the enzyme activity of the bacterium di¡ered from other prokaryotic and eukaryotic arginases in its activation by divalent cations. Mn2‡ is the most potent stimulator of arginases from many sources including plants [40], yeast [41], fungi [36], and bacteria [24,26,27,29]. Mammalian liver arginases are also manganoproteins, and Mn2‡ is thought to be their physiological activator [34,42,43]. Divalent cations stimulated H. pylori arginase activity in situ and

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in semi-puri¢ed enzyme preparations, but signi¢cantly larger e¡ects were observed with Co2‡ or Ni2‡ than with Mn2‡ (Table 2). In contrast, Co2‡ inhibited B. anthracis and R. capsulatum E1F1 arginases, and Ni2‡ inhibited the former and did not a¡ect the latter [24,29]. Human hepatic arginase is also activated by Ni2‡ and Co2‡ , but the order of activation was also Mn2‡ s Ni2‡ s Co2‡ [2]. In common with other bacterial arginases, the H. pylori enzyme was inhibited by Cu2‡ or Zn2‡ [24,29,44]. Rat and mouse liver arginases require added divalent cations for activity, but are not inhibited by metal chelators such as EDTA [34,45]. The H. pylori enzyme activity was measured in lysates without the addition of divalent cations, suggesting that metal ions remained bound to the enzyme even after considerable dilution, but activity was susceptible to the presence of EDTA (Table 4). The high speci¢city of H. pylori arginase activity was established by observing in situ the metabolism of L-arginine analogs (Table 3). The enzyme recognized only the L-stereoisomer and, with the exception of L-argininamide, did not catabolize metabolites with modi¢ed amino, carboxylic or guanidino groups. Substrates with relatively small changes in the chain length, such as L-canavanine or L-homoarginine, were not catabolized by H. pylori, in contrast with B. caldovelox [26] and some eukaryotic liver arginases which hydrolyze both metabolites at very slow rates [2,34,46]. In the case of L-canavanine, it should be noted also that at neutral pH a signi¢cant proportion of the guanidinium group will be deprotonated (pK 6.6), whereas the guanidinium group of L-arginine (pK 12.5) will be protonated. Although Lcanavanine and L-homoarginine were not hydrolyzed by H. pylori, their presence interfered with the hydrolysis of arginine (Table 3), suggesting stringent steric and geometrical constrains at the active site. The arginases of B. anthracis, B. caldovelox, and R. capsulatum E1F1 are less speci¢c than that of H. pylori, as the former catabolize various arginine analogs and also show activity with L-ornithine [24,26,29]. In contrast, B. subtilis arginase is inhibited by L-ornithine [44]. Rat liver arginase also is inhibited by L-ornithine and catabolizes L-arginine analogs with modi¢cations in the end groups or side chain such as those in Table 3 [34]. These results indicated signi¢cant di¡erences between the speci¢city of the

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H. pylori enzyme and other bacterial and mammalian liver arginases. The H. pylori arginase activity was inhibited by the mercurial p-chloromercuribenzoate, like those of B. anthracis [29] and R. capsulatus [24], and the alkylating agent iodoacetamide, suggesting that sulfhydryl groups are important for activity although they may not be involved in catalysis directly. Five of the six cysteinyls in H. pylori arginase are not present in any of the other 16 arginase sequences [3], and only Cys312 is conserved in the sequences of the eukaryote enzymes from mammalian liver, Rana catesbeiana, Xenopus laevis and S. pombe, but not in any of the prokaryote arginases. Inhibition of arginase by urea was reported only for the ¢rst time for the rat hepatic arginase [34]. The lack of data on urea inhibition is due, in part, to the fact that most assays quantitate the urea produced in the reaction. The H. pylori enzyme was inhibited by urea with a Ki that was an order of magnitude smaller than that of rat type I arginase. Approximately 50% of the arginase activity measured in lysates prepared from growing H. pylori cultures remained in lysates prepared from suspensions with coccoid forms of the bacterium, approximately 30% of the original enzyme activity was present in the catalytically active complex, suggesting that this enzyme may play a role in the survival of the bacterium. The e¡ects of bicarbonate on arginase activity (Fig. 3) indicated that the enhancement of activity observed in cells did not arise from an intrinsic property of the enzyme nor from modulation of arginine transport into the cells by the anion, but was a property of arginase in situ. The strict requirement for 3^ 10% CO2 levels in the growth atmosphere of H. pylori cultures in vitro is one of the microaerophilic properties of the bacterium. Since urea is one of the catalytic products of arginase, the enzyme participates in the nitrogen metabolism of H. pylori through its role in the urea cycle [12]. A characteristic feature of the physiology of the bacterium is the production of copious amounts of very active urease, and hydrolysis of urea yields ammonium and bicarbonate. Consequently, arginase may be implicated also in the microaerophily of the bacterium owing to the modulation of its activity by bicarbonate. The enzyme would be a point of contact between

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nitrogen metabolism and the microaerophily of H. pylori, two fundamental areas of the physiology of this microorganism. The sequence of H. pylori arginase showed a low degree of similarity with other known arginases (Table 5; [3]), and amongst the closer sequences were those of B. subtilis and mammalian arginases. Mammalian hepatic and non-hepatic arginases have similar reaction kinetics, divalent cation requirements and quaternary structure. However, in terms of size and subcellular location, isoelectric point, immunological cross-reactivity and charge, signi¢cant di¡erences exist between these enzymes. The deduced amino acid sequence of H. pylori arginase indicated that it had a similar size to type II arginases (Table 5), and the data on enzyme location suggested also similarities with these mammalian isozymes. The calculated isoelectric point of the H. pylori enzyme was closer to those of type II arginases than to the pI of the other microbial or mammalian type I enzymes (Table 5). There were similarities between the physicochemical properties of H. pylori arginase activity and those of B. subtilis and mammalian arginases, but signi¢cant functional di¡erences in the activation by divalent cations, speci¢city, and e¡ects of inhibitors, may re£ect the uniqueness of the H. pylori enzyme. The presence of a 13-residue segment as an insertion not found in any other arginase supported this conclusion about H. pylori arginase. Experiments are under way to elucidate the role of this segment. Acknowledgements This work was made possible by the support of the Australian Research Council and the National Health and Medical Research Council of Australia. We are grateful for the help of the Baxter Perpetual Charitable Trust. References [1] C.A. Ouzonis, N.C. Kyrpides, J. Mol. Evol. 39 (1994) 101^ 104. [2] N. Carvajal, C. Torres, E. Uribe, E. Salas, Comp. Biochem. Physiol. 112B (1995) 153^159.

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