Agmatine oxidation by copper amine oxidase Biosynthesis and biochemical characterization of N -amidino-2-hydroxypyrrolidine Paolo Ascenzi 1, *, Mauro Fasano 2, *, Maria Marino 1 , Giorgio Venturini 1 and Rodolfo Federico 1 1 Department of Biology, University ÔRoma TreÕ, Rome, Italy; 2 Department of Structural and Functional Biology, University of Insubria, Varese, Italy The p roduct of agmatine oxidation catalyzed by Pisum sativum L. copper amine oxidase has been identified by means of one- and two-dimensional 1 H-NMR spectroscopy to be N-amidino-2-hydroxypyrrolidine. This compound inhibits competitively rat nitric oxide synthase type I and type II (NOS-I and NOS-II, respectively) and bovin e t rypsin (trypsin) activity, values of K i being (1.1 ± 0.1) · 10 )5 M (at pH 7.5 and 37.0 °C), (2.1 ± 0.1) · 10 )5 M (at pH 7.5 and 37.0 °C), and (8.9 ± 0.4) · 10 )5 M (at p H 6.8 and 21.0 °C), respectively. Remarkably, the affinity o f N-amidino- 2-hydroxypyrrolidine f or NOS-I, NOS-II and trypsin is significantly higher than that observed for agmatine and clonidine binding. Furthermore, N-amidino-2-hydroxy- pyrrolidine a nd agmatine are more efficient than clonidine in displacing [ 3 H]clonidine (¼ 1.0 · 10 )8 M ) from specific binding sites in heart rat membranes, values of IC 50 being (1.3 ± 0.4) · 10 )9 M and (2.2 ± 0.4) · 10 )8 M ,respec- tively (at pH 7.4 an d 37.0 °C). Keywords: c opper amine oxidase; agmatine; N-amidino-2- hydroxypyrrolidine; enzyme inhibition; type 1 imidazoline receptor b ind ing. Copper a mine oxidase has been identified in b acteria, yeasts, fungi, plants, a nd animals. This enzyme is a homodimer o f 70- to 90-kDa subunits, each c ontaining a s ingle copper i on and a covalently bound cofactor formed by the post- translational modification of the catalytic tyrosyl residue to 2, 4,5-trihydroxyphenylalanine quinone (TPQ) [ 1–4]. Copper amine oxidase catalyzes the oxidative deamination of biogenic amines, including mono, di, and polyamines, neurotransmitters such as catecholamines, histamine and xenobiotic amines, with substrate p references depending upon the enzyme source [1–5]. The copper amine oxidase catalyzed reactions follow t he general s cheme: E ox þ R-CH 2 -NH 2 ! E red þ R-CHO ðreaction 1Þ E red þ O 2 þ H 2 O ! E ox þ NH 3 þ H 2 O 2 ðreaction 2Þ where E ox represents the enzyme–quinone, R-CH 2 -NH 2 is the substrate, E red is the enzyme–aminoquinol, and R-CHO is the product aldehyde. Substrate amines interact directly with TPQ in the reductive part of the process forming a Schiff base complex (reaction 1). Proton abstraction of the substrate, catalyze d by an invariant Asp residue, leads to the release of product aldehyde and leaves the enzyme in the reduced aminoquinol form (reaction 1) [1–4]. The oxidative part (reaction 2) leads to reoxidation of the aminoquinol cofactor with the release of ammonia and hydrogen peroxide [1–4]. Copper amine oxidase catalyzes also the oxidation of agmatine [3–5], which has been recognized to be an impor- tant bioactive molecule, b eing identified as a novel neuro- transmitter and modulator of cardiovascular functions via binding to type 1 imidazoline (I 1 -R) and a-adrenergic receptors [6,7]. Interestingly, agmatine inhibits nitric oxide synthase isoforms [8,9] and induces the release of some peptide hormones [7]. To date, the product(s) of the copper amine oxidase catalyzed oxidation of agmatine has not been identified. Moreover, no information is available o n the role played by the product(s) of agmatine metabolism on cell function(s). Here, the b iosynthesis and the biochemical characterization of N-amidino-2-hydroxypyrrolidine, the product of agmatine oxidation by Pisum sativum L. copper amine oxidase, is reported. MATERIALS AND METHODS Proteins P. sativum copper amine oxidase was purified as previously reported [10]. Rat nitric oxide synthase type I (NOS-I) was prepared from the rat brain homogenate [11]. Rat nitric oxide synthase type II (NOS-II) was prepared from the lung homogenate of rats treated with E. coli lipopolysaccharide (10 mgÆkg )1 ) [11]. NOS-I and NOS-II containing specimens were homogenized at pH 7.5 (5.0 · 10 )2 M Hepes buffer), 5.0 · 10 )4 M EGTA, 1.0 · 10 )3 M dithiothreitol, and 0.1 mgÆmL )1 phenylmethanesulfonyl fluoride [11]. Then, Correspondence to P. Ascenzi, Dipartimento di Biologia, Universita ` ÔRoma TreÕ, Viale Guglielmo Marconi 446, I-00146 Rome, Italy. Fax: + 39 06 551 76321, Tel.: + 39 06 55176329, E-mail: ascenzi@uniroma3.it Abbreviations:I 1 -R, type 1 imidazoline receptor; MMFF, Merck Molecular Force Field; NOS-I, rat nitric oxide synthase type I (neu- ronal constitutive isoform); NOS-II, rat nitric oxide synthase type II (inducible isoform); TPQ, 2,4,5-trihydroxyphenylalanine quinone; trypsin, bovine trypsin. Enzymes: bovine catalase (EC 1.11.1.6); bovine trypsin (EC 3.4.21.4); Pisum sativum L. copper amine oxidase (EC 1.4.3.6); rat nitric oxide synthase type I (EC 1.14.13.39); rat nitric oxide synthase type II (EC 1.14.13.39). *Note: These authors contributed equally to this work. (Received 26 July 2 001, r evised 1 7 October 2001, acc epted 3 December 2001) Eur. J. Biochem. 269, 884–892 (2002) Ó FEBS 2002 NOS-I and NOS-II containing homogenates were desalted by chromatography over disposab le PD-10 columns packed withSephadexG-25medium(AmershamPharmaciaBio- tech, Uppsala, Sweden). Bovine calmodulin, bovine cata- lase, bovine serum albumin, bovine trypsin (trypsin), and horseradish peroxidase were purchased from Sigma Chemical Co (St Louis, MO, USA). Proteins were of reagent grade and used without further purification. Chemicals Agmatine, aminoantipyrine, N-a-benzoyl- L -arginine p-nitro- anilide, clonidine, 3,5-dichloro-2-hydroxybenzenesulfonic acid, epinephrine, phenylmethanesulfonyl fluoride, and Escherichia coli lipopolysaccharide (serotype 0127:B8) were obtained from Sigma Chemical Co. [ 3 H] L -arginine (specific activity 2.0 TBqÆmmol )1 )and[ 3 H]clonidine (specific activity 2.6 TBqÆmmo l )1 ) were purchased from NEN TM Life Science Products (Boston, MA, USA). Deuterium oxide (99.8% isotopic enrichment) was obtained f rom C ortec (Paris, France). All the other chemicals were from Merck AG (Darmstadt, Germany). All products were of analytical or reagent grade and u sed without further purification. Animals Male Sprague–Dawley rats (from Morini, I taly), 4- to 5-month-old, were housedandacclimatized for 1 week un der controlled temperature (20 ± 1 °C), humidity (55 ± 10%), and light (from 7 a.m. to 7 p.m) conditions. T he rats were anaesthetized with ether in a fume hood, and o rgans removed and rapidly chilled in liquid nitrogen (brain and lung) or i n ice-cold medium solution (2.0 · 10 )2 M NaHCO 3 ; heart). Animal experiments were p erformed accor- ding to ethical guidelines for t he conduct o f animal r esearch. P. sativum copper amine oxidase assay Oxidation of agmatine by P. sativu m copper amine oxi- dase was investigated s pectrophotometrically by follo- wing the formation of a pink adduct ( e 515nm ¼ 2.6 · 10 4 M )1 Æcm )1 ), as a result of the oxidation of aminoanti- pyrine and 3,5-dichloro-2-hydroxybenzenesulfonic acid cat- alyzed by horseradish peroxidase, at pH 7.0 (1.0 · 10 )1 M phosphate buffer) and 25.0 °C [5,6,10]. In a typical experi- ment, 20 lL of a buffered P. sativum copper amine oxidase solution (1.0 · 10 )1 M phosphate buffer, pH 7.0) wereaddedtoabufferedsolution(1.0mL;1.0· 10 )1 M phosphate buffer, pH 7.0) containing the substrate (i.e. agmatine), aminoantipyrine ( 1.0 · 10 )4 M ), 3 ,5-dichloro- 2-hydroxybenzenesulfonic acid (1.0 · 10 )3 M ), and horse- radish peroxidase (1.5 · 10 )6 M ). The initial velocity for the enzymatic oxidation of agmatine was then measured. P. sativum copper amine oxidase activity was also assayed polarographically with a Clark electrode (Hansa- tech Instruments Ltd, Norfolk, UK) by following the O 2 consumption, at pH 7.0 (1.0 · 10 )1 M phosphate buffer) and 25.0 °C [12]. In a typical experiment, 20 lLofa buffered agmatine solution (1.0 · 10 )1 M phosphate buffer, pH 7.0) were added to a buffered s olution (1.0 mL; 1.0 · 10 )1 M phosphate buffer, pH 7.0) containing P. sativum copper amine oxidase. The initial velocity for the enzymatic oxidation of agmatine was then measured. In the e nzyme assay, t he P. sativum copper amine oxidase concentration was 5.0 · 10 )9 M and the agmatine concen- tration ranged between 5.0 · 10 )5 M and 5.0 · 10 )3 M .The enzyme activity was linear up to 5 min of incubation and results were expressed as lmol productÆs )1 Æ(lmo l e nzy me) )1 . Under all th e e xperimental c onditions, t he initial velocity for the P. sativum copper amine oxidase catalyzed oxidation of agmatine was unaffected by the enzyme/substrate incuba- tion time. In fact, the enzyme/substrate equilibration time was very short, being completed within the mixing time (% 15 s). Values of the first-order rate-limiting catalytic constant (k cat ) and of the Michaelis constant, as determined in the absence of the inhibitor (K 0 m )fortheP. sativum copper amine oxidase catalyzed oxidation of agmatine, were obtained from the dependence of the initial velocity for agmatine oxidation ( v i ) on the substrate (i.e. agmatine) concentration ([S]), according to Eqn (1) [13]: v i ¼ k cat ½S=ðK 0 m þ½SÞ ð1Þ Values of k cat and K 0 m for the P. sativum copper amine oxidase catalyzed oxidation of agmatine are 1.3 ± 0.1 s )1 and (3.8 ± 0.3) · 10 )4 M , r espectively, at pH 7.0 ( 1.0 · 10 )1 M phosphate buffer) and 25.0 °C (Fig. 1). Values of k cat and K 0 m are independent of the enzyme assay. Biosynthesis of N -amidino-2-hydroxypyrrolidine N-Amidino-2-hydroxypyrrolidine was synthesized as fol- lows. Twenty micrograms of P. sativum copper a mine oxidase were added to 1.0 mL of a buffered 2.0 · 10 )3 M agmatine solution (5.0 · 10 )2 M phosphate buffer, pH 7.4). 28 lg of bovine catalase were also added to the reaction solution (1.0 mL) in order to remove H 2 O 2 , arising from the P. sativum copper a mine oxidase catalyzed oxidation of agmatine. The reaction solution was stirred vigorously at 25.0 °C for 20 min, and the product recovered by ultrafil- tration on Amicon P M10 membranes (Amicon, Inc., Beverly, MA, USA). Fig. 1. Effect of substrate (i.e. agmatine) concentration on values of v i for the P. sativ um copper amine oxidase catalyzed oxidation of agma- tine. T he c ontinuou s line was calculated according t o Eqn (1), with the following values of k cat (¼ 1.3±0.1s )1 )andK 0 m [¼ (3.8 ± 0.3) · 10 )4 M ]. Data were obtained at pH 7.0 a nd 25.0 °C, mean ± SD. Fo r further details, see text. Ó FEBS 2002 N-Amidino-2-hydroxypyrrolidine characterization (Eur. J. Biochem. 269) 885 The total conversion of agmatine to N-amidino-2- hydroxypyrrolidine was detected by 1 H-NMR s pectroscopy. Moreover, the agmatine/N-amidino-2-hydroxypyrrolidine stoichiometry is 1 : 1 as shown by 1 H-NMR spectroscopy. The N-amidino-2-hydroxypyrrolidine concentration was determined from 100% conversion of agmatine to N-amidino-2-hydroxypyrrolidine a s d emonstrated by 1 H-NMR spectroscopy. Under a ll the experimental conditions, the formation of free 4-guanidinobutyraldehyde was observed n either by the o-aminobenzaldehyde assay [14] (data not shown) nor 1 H-NMR spectroscopy (Figs 2 and 3). NMR spectroscopy P. sativum copper amine oxidase catalyzed oxidation of agmatine was conducted as described above, in deuterated phosphate buffer (pD 7 .4; uncorrected pH-meter reading 7.0); residual oxygen was removed with a mild nitrogen stream. A control s pectrum was recorded prior to addition of P. sativum copper amine oxidase. 1 H-NMR one- and two-dimensional spectra were recorded at 25.0 °Cona Bruker AVANCE 600 NMR spectrometer (Bruker Ana- lytik, Rheinstetten, Germany), operating at a magnetic field strength of 14.1 T. The residual water signal was s uppressed by a 2-s presaturation before the observation pulse. The duration of the pulse corresponding to a flip angle of 90° was 7 .4 ls. The spin system o f the agmatine oxidation product was assigned by COSY, by setting the flip angle of the second pulse to 35°. T o this purpose, 256 t 1 increments were recorded (4096 points each). The resulting matrix was zero-filled to 1024 · 4096 complex points and processed with a 5 °-shifted squared sinebell in both dimensions [15]. Building of the N -amidino-2-hydroxypyrrolidine structure Energy minimization of the proposed structure of N-amidino-2-hydroxypyrrolidine w as performed on a Silicon Graphics Octane workstation (SGI, Mountain View, CA, USA) by using the program SPARTAN (Wave- function Inc., Irvine, CA, USA). NOS-I and NOS-II assay NOS-I and NOS-II activity was assessed by evaluating the conversion of [ 3 H] L -arginine to [ 3 H] L -citrulline at pH 7.5 (5.0 · 10 )2 M Hepes buffer) and 37.0 °C, in the absence and presence of N-amidino-2-hydroxypyrrolidine. In a typical experiment, a NOS-I or NOS-II aliquot (50 lL) was a dded to the reaction mixture (100 lL) containing 1.0 · 10 )3 M NADPH, 1.2 · 10 )3 M CaCl 2 ,1.0lgÆmL )1 calmodulin, 1.0 · 10 )5 M FAD, 1.0 · 10 )5 M FMN, [ 3 H] L -arginine (from 12 to 185 kBq) and L -arginine (from 1.0 · 10 )6 M to 1. 0 · 10 )4 M ), in the absence and presence of N-ami- dino-2-hydroxypyrrolidine (from 5.0 · 10 )6 M and 5.0 · 10 )5 M ). For the determination of NOS-II activity, CaCl 2 and calmodulin were omitted, and 1.0 · 10 )3 M EGTA was added to the reaction mixture. NOS-I and NOS-II activity was assayed in the presence of 5.0 · 10 )5 M BH 4 [16]. In the enzyme assay, the NOS-I or NOS-II concentration was 2.0 · 10 )7 M . After 15 min incubation, the reaction was stopped by addition of an ice-cold 2.0 · 10 )2 M Hepes buffer solution (700 lL), pH 5.5, containing 2.0 · 10 )3 M EDTA. [ 3 H] L -citrulline was separated from [ 3 H] L -arginine by ion exchange chromatography on Dowex 50WX8 (Fluka Chemie AG) [11,16]. The enzyme activity was linear up to 30 min of incubation and results were expressed as pmol productÆmin )1 Æ(mg protein) )1 . U nder all the experimental conditions, the initial velocity for NOS-I a nd NOS-II catalyzed conversion of L -arginine to L -citrulline was unaffected by the enzyme/inhibitor/substrate incuba- tion time. In fact, the enzyme/inhibitor/substrate equilibra- tion time was very short, being completed within the mixing time (% 15 s). Values of the first-order rate-limiting catalytic constant (k cat ) and of the Michaelis constant, as determined in the absence and presence of the inhibitor (K 0 m and K app m , respectively), for NOS-I and NOS-II catalyzed conversion of L -arginine to L -citrulline were obtaine d from the depen- dence of the initial velocity for substrate conversion ( v i )on the L -arginine concentration ([S]), according to Eqn (1) [13]. Values of k cat and K 0 m for the NOS-I catalyzed conversion of L -arginine to L -citrulline were 1 .4 ± 10 2 pmol prod- uctÆmin )1 Æ(mg protein) )1 and 4.0 · 10 )6 M , respectively, at pH 7.5 and 37.0 °C [11]. Values of k cat and K 0 m for the NOS-II catalyzed conversion of L -arginine to L -citrulline were 4.7 · 10 1 pmol productÆmin )1 Æ(mg protein) )1 and 1.8 · 10 )5 M , respectively, at pH 7.5 and 37.0 °C [17]. NO production was also monitored spectrophotometri- cally (between 350 and 460 nm) following the NO-mediated conversion of human oxy-hemoglobin (6.0 · 10 )6 M ), added to the N OS-I and NOS-II preparations, to m et-hemoglobin, Fig. 2. 1 H-NMR spectra of 2.0 · 10 )3 M agmatine before (A) and after (B) oxidation catalyzed by P. sativum copper amine oxidase, at pD 7.4 and 25.0 °C. Acquisition param- eters: 4 scans, flip angle 45°, relaxation delay 2 s. T he residual water s ignal was suppressed by presaturation. For further details, see text. 886 P. Ascenzi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 in the presence of N-amidino-2-hydroxypyrrolidine as the substrate instead of L -arginine, at pH 7.5 ( 5.0 · 10 )2 M Hepes buffer) and 37.0 °C [18,19]. Trypsin assay The trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroanilide was investigated spectrophotometrically (at 408 nm), at pH 6.8 (1.0 · 10 )1 M phosphate buffer) and 21.0 °C [20], in the absence and presence of N-amidino- 2-hydroxypyrrolidine. In a typical experiment, 20 lLofa buffered trypsin solution (1.0 · 10 )1 M phosphate buffer, pH 6.8) were added to 1.0 mL of a buffered solution (1.0 · 10 )1 M phosphate buffer, pH 6.8) containing the substrate (i.e. N-a-benzoyl- L -arginine p-nitroanilide) and the inhibitor (i.e. N-amidino-2-hydro xypyrrolidine). The initial velocity for the enzymatic hydrolysis of N-a-benzoyl- L -arginine p-nitroanilide was then measured. In the enzyme assay, the trypsin concentration was 1.0 · 10 )6 M ,the N-a-benzoyl- L -arginine p-nitroanilide concentration ranged between 1.0 · 10 )5 M and 1.0 · 10 )3 M ,andtheN-ami- dino-2-hydroxypyrrolidine concentration ranged between 2.0 · 10 )5 M and 8.0 · 10 )5 M . The enzyme activity was linear up to 10 min of incubation an d results were expressed as lmol productÆs )1 Æ(lmol enzyme) )1 .Underalltheexper- imental conditions, the initial velocity for the trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroani- lide w as unaffected by the enzyme/inhibitor/substrate incubation time. In f act, the enzyme/inhibitor/substrate equilibration time was very short, being completed within the mixing time (% 15 s). Values of the first-order rate-limiting catalytic constant (k cat ) a nd of the Michaelis constant determined in the absence and presence of the inhibitor (K 0 m and K app m , respectively) for the trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroanilide were obtained from the d ependence of the initial velocity f or substrate hydrolysis (v i )ontheN-a-benzoyl- L -arginine p-nitroani- lide c oncentration ([S]), according to Eqn (1) [13]. Values of k cat and K 0 m for the trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-n itroanilide were 0.70 s )1 and 3.0 · 10 )4 M , respectively, at pH 6.8 and 21.0 °C[20]. Determination of values of the inhibition dissociation equilibrium constant ( K i ) for N -amidino-2-hydroxypyrrolidine binding to NOS-I, NOS-II, and trypsin Values of the inhibition dissociation equilibrium constant (K i ) for the competitive inhibition of the N OS-I and NOS-II catalyzed conversion of L -arginine to L -citrulline (at pH 7.5 and 37.0 °C) and of the trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroanilide (at pH 6.8 and 21.0 °C) by N-amidino-2-hydroxypyrrolidine were deter- mined from the linear dependence of the K app m /K 0 m ratio on the inhibitor concentration (i.e. [I]), according to Eqn (2) [13]: K app m =K 0 m ¼ K À 1 i ½Iþ1 ð2Þ As expected for a simple competitive inhibition system [13], values of k cat for the NOS-I a nd NOS-II catalyzed conversion of L -arginine to L -citrulline and for the trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroani- lide were unaffected by the inhibitor concentration within the standard deviation (± 5%). Model building of the NOS-II: and trypsin: N -amidino-2-hydroxypyrrolidine complexes Molecular models of the human NOS-II: and bovine trypsin:N-amidino-2-hydroxypyrrolidine complexes were built using the coordinates of the human NOS-II:S-ethyl- Fig. 3. Two-dimensional COSY spectrum of N-amidino-2-hydroxy- pyrrolidine, the cyclic oxidation product of agmatine, at pD 7.4 and 25.0 °C (top) and ball-and-stick model of N-amidino-2-hydroxypyrro- lidine (bottom). Acquisition parameters: 4 scans, 1 6 dummy scans, relaxation delay 2 s. Labels refer to the resonance assignment in Fig. 1B. For further details see text. Ó FEBS 2002 N-Amidino-2-hydroxypyrrolidine characterization (Eur. J. Biochem. 269) 887 isothiourea complex (PDB accession no. 4NOS) [21] and the bovine t rypsin:benzamidine adduct (PDB a ccession no. 1CE5) [22] as templates, respectively. The atomic coordi- nates o f rat NOS-II are not yet ava ilable [23], the homologous human enzyme was used instead. The confor- mations of the N-amidino-2-hydroxypyrrolidine in the enzyme:inhibitor complexes were obtained after 10 ps molecular dynamics. Ene rgy minimization and molecu lar dynamics were performed on a Silicon Graphics O 2 workstation ( SGI, Irvine, CA, USA) with HYPERCHEM 4.5 for SGI (Hypercube Inc., Gainesville, FL, USA). I 1 -R binding assay Cardiac muscle (cleaned of connective t issue and fat) was finely minced and homogenized in ice-cold medium solution 2.0 · 10 )2 M NaHCO 3 , c ontaining 1.0 · 10 )4 M phen- ylmethanesulfonyl fluoride, with a wet weight to volume ratio of 1 : 7, using a glass-Teflon homogenizer (10 · 30 s) [24]. The homogenate was centrifuged at 1500 g for 15 min (4.0 °C). The supernatant was centrifuged at 45 000 g for 5 min (at 4.0 °C). The pellet was washed twice, then re-suspended i n 2 mL of ice-cold 5.0 · 10 )3 M Hepes buffer, containing 5.0 · 10 )4 M EGTA, 5.0 · 10 )4 M MgCl 2 ,and 1.0 · 10 )4 M ascorbic acid (pH 7.4) [25]. Membrane pre- parations were f ree o f m itochondria and nuclei as confirmed by subcellular enzymatic marker assays (data not shown). Two-hundred and forty micrograms of membrane pro- tein were incubated for 55 min with 1.3 nmol to 40 nmol [ 3 H]clonidine at 37.0 °C in a final volume of 0.5 mL of 5.0 · 10 )3 M Hepes buffer, con taining 5.0 · 10 )4 M EGTA, 5.0 · 10 )4 M MgCl 2 ,and1.0· 10 )4 M ascorbic acid (pH 7 .4). The reaction was stopped by rapid vacuum filtration with a Millipore harvester throughWhatman GF/C glass fiber filters (Whatman International Ltd Maidstone, UK) p resoaked with 10% polyethyleneglycol in Tris/HCl 2.0 · 10 )2 M , containing MgCl 2 1.0 · 10 )2 M , followed by rapid washing of filters with 10 mL ice-cold 5.0 · 10 )3 M Hepes buffer, containing 5.0 · 10 )4 M EGTA, 5.0 · 10 )4 M MgCl 2 ,and1.0· 10 )4 M ascorbic acid (pH 7.4). Filters were placed in a 6-mL scintillation fluid and the radio- activity determined by liquid s cintillation counting. Epine- phrine (1.0 · 10 )5 M ), which does not bind to imidazoline sites [26,27], was added to the assay to prevent [ 3 H]clonidine from binding to a-adrenergic receptors. Nonspecific binding wasdefinedas[ 3 H]clonidine-binding (the [ 3 H]clonidine concentration ranged between 1.5 · 10 )4 M and 5.0 · 10 )4 M ). Saturation studies were performed with 1.0 · 10 )8 M [ 3 H]clonidine and increasing concentrations of the unl abelled ligand (i.e. N-amidino-2-hydroxypyrroli- dine, agmatine, and clonidine; f rom 1.0 · 10 )9 M to 1.0 · 10 )6 M ). Protein concentration was measured by the method of Bradford [28], using bovine serum albumin as the standa rd. Values of IC 50 for [ 3 H]clonidine displacement from I 1 -R in heart rat membranes by N-amidino-2-hydroxypyrroli- dine, agmatine, and clonidine were determined according to Eqn (3): a ¼ 1=f1 þð½L=IC 50 Þg ð3Þ where a is the m olar fraction of [ 3 H]clonidine bound to I 1 -R present in heart rat membranes and [L] is the concentration of the ligand (i.e. N-amidino-2-hydroxypyrrolidine, agma- tine, or clonidine) [29]. RESULTS Over the w hole substrate ( i.e. agmatine) concentration range explored (i.e. between 5.0 · 10 )5 M and 5.0 · 10 )3 M ), the P. sativum copper amine oxidase cata- lyzed oxidation of agmatine follows simple Michaelis– Menten kinetics (Fig. 1). According to the literature [30], values of k cat and K 0 m for the P. sativum copper amine oxidase catalyzed oxidation of agmatine are 1.3 ± 0.1 s )1 and (3.8 ± 0.3) · 10 )4 M , respectively, at pH 7.0 and 25.0 °C. Moreover, values of k cat and K 0 m were independent of the enzymatic assay used (spectrophotometric vs. pola- rographic). The stoichiometric analysis of the enzymatic oxidation of agmatine yields a molar ratio of sub strate (i.e. agmatine) to O 2 and H 2 O 2 of 1 : 1 : 1. Figure 2 shows the 1 H-NMR s pectra of agmatine before (Fig. 2 A) and after (Fig. 2B) oxidation catalyzed by P. sativum copper amine oxidase, at pD 7.4 and 25.0 °C.Theagmatinesampleshowssomesignalsatthe impurity level, which however do not hamper the observation of the main component. The main features of Fig. 2B with respect to Fig. 2A are: (a) the upset of a downfield-shifted signal at d ¼ 5.5 p.p.m., and (b) the splitting of CH 2 signals in magnetically unequivalent components. On the basis of the general mechanism (see reactions 1 and 2), one trip let (relative area 1) should occur at about d ¼ 9 p.p.m., corresponding to the formyl proton, one triplet at about d ¼ 3 p.p.m. (relative area 2), and two multiplets at about d ¼ 2 p.p.m. (relative area 2 each). As the -CHO signal w as not observed, the formation of the corresponding free aldehyde (i.e. 4- guanidobutyraldehyde) was ruled out. To note t hat the agmatine/N-amidino-2-hydroxypyrrolidine stoichiometry is 1 : 1 as shown by 1 H-NMR spectroscopy. A possible explanation for the resoluti on of the magnetic equivalence of CH 2 groups would be the formation of an intramolecular Sc hiff base in its emiac- etalic form, deriving from nucleophilic attack of the guanidinic e N nitrogen to the (transient) aldehydic carbonyl. T his implies the formation of a chiral center on the ring, with all CH 2 protons consequently becoming diastereotopic and hence magnetically non equivalent (see Scheme 1). As the presence of free 4-guanidobutyralde- hyde was never detected, the formation of the cyclic product N-amidino-2-hydroxypyrrolidine should o ccur within the enzyme catalytic center (shown within square brackets in Scheme 1). Figure 3 (top panel) shows the magnitude COSY spec- trum of the product of agmatine oxidation catalyzed by P. sativum copper amine oxidase. Starting from the emiac- etalic proton A, it is possible to walk over the whole spin system and identify the connectivities on the basis of 3 J scalar couplings [15]. As three-bond couplings were not observed, it was assumed that the involved protons form dihedral angles close to 90° [31]. I n other word s, the absence of scalar coupling between A a nd, say, C identified the axial- equatorial pairs. Figure 3 (bottom panel) shows the ball- and-stick model of N-amidino-2-hydroxypyrrolidine (the product of agmatine o xidation catalyzed by P. sativum copper amine oxidase) after 200 cycles of energy minimi- 888 P. Ascenzi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 zation in the MMFF force field [32], with torsion angles constrained according to the results of the COSY spectrum (Fig. 3, top panel). AsshowninFig.4,N-amidino-2-hydroxypyrrolidine inhibits competitively t he NOS-I and NOS-II catalyzed conversion of L -arginine to L -citrulline and the trypsin catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroani- lide. Table 1 gives K i values for N-amidino-2-hydroxy- pyrrolidine (present study), agmatine [8,30], and clonidine [16,30] binding to NOS-I, NOS-II, and trypsin. Remark- ably, the affinity of N-amidino-2-hydroxypyrrolidine for NOS-I, NOS-II, and trypsin is systematically higher than that observed for agmatine and clonidine b inding (see Table 1 ). As reported for agmatine [8] an d clonidine [16], N-amidino-2-hydroxypyrrolidine is not a NO precursor. In fact, human oxy-hemoglobin added to NOS-I and NOS-II preparations is not converted to met-hemoglobin in the presence of N-amidino-2-hydroxypyrrolidine as t he sub- strate instead of L -arginine (data not shown). Figure 5 shows the molecular models of the human NOS-II: and bovine trypsin:N-amidino-2-hydroxypyrro- Scheme 1. Fig. 4. Effect of N-amidino-2-hydroxypyrrolidine c oncentration (i.e. [Inhibitor]) on the K app m =K 0 m ratio for the competitive inhibition of NOS-I (squares) and NOS-II (triangles) catalyzed conversion of L -arginine to L -citrulline, and of the trypsin (circles) c atalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroanilide. The con tinuous lines were cal- culated according to Eqn (2) with values of K i giveninTable1.Data were obtained between pH 6.8 and 7.5 and between 21.0 °Cand 37.0 °C, mean ± SD, for further details, s ee text. Table 1. Values of K i for N-amidino-2-hydroxypyrrolidine, agmatine, and clonidine binding to NOS-I, NOS-II, and trypsin. Enzyme K i ( M ) N-Amidino-2- hydroxypyrrolidine Agmatine Clonidine NOS-I (1.1 ± 0.1) · 10 )5a (6.6 ± 1.1) · 10 )4b (5.0 ± 0.2) · 10 )3c NOS-II (2.1 ± 0.1) · 10 )5a (2.2 ± 0.2) · 10 )4b >5 · 10 )2c Trypsin (8.9 ± 0.4) · 10 )5d >10 )2e >10 )2e a pH 7.5 and 37.0 °C. Present study. b pH 7.8 and 37.0 °C. From [8]. c pH 7.5 and 37.0 °C. From [16]. d pH 6.8 and 21.0 °C. Present study. e pH 7.0 and 25.0 °C. From [30]. Ó FEBS 2002 N-Amidino-2-hydroxypyrrolidine characterization (Eur. J. Biochem. 269) 889 lidine co mplexes. I n human NOS-II (top panel), N-amidino- 2-hydroxypyrrolidine is hosted in the hydrophobic cavity defined by the heme p rosthetic group and by the facing hydrophobic residues Ala270 and Val271, as observed for a number of nitrogen heterocycles [21,33] (note that N-ami- dino-2-hydroxypyrrolidine is constrained in a semiboot conformation, with the nitrogen lone pair directed towards the heme iron). The positively charged amidino group of N-amidino-2-hydroxypyrrolidine appears to be stabilized by the negatively charged carboxylate of the Glu296 residue which is required for L -arginine binding [21,33]. By homo- logy, this residue corresponds to Glu597 and Glu371 in rat NOS-I and NOS-II, respectively [23]. Moreover, as previ- ously reported for the bovine trypsin:benzamidine complex [22,34], N-amidino-2-hydroxypyrrolidine binds to the enzyme primary s pecificity subsite S 1 (bottom p anel). Interestingly, the alicyclic group is extended in a se michair conformation, with the positively charged amidino g roup of N-amidino-2-hydroxyp yrrolidine forming a salt bridge with the negatively charged carboxylate of the trypsin Asp189 residue. The latter is required for recognition of the cationic amino acid residue present at t he P 1 position of substrates and inhibitors of trypsin-like serine proteinases [35,36]. N-Amidino-2-hydroxypyrrolidine, agmatine, and cloni- dine bind to I 1 -binding sites (i.e. I 1 -R). In fact, the I 2 sites, which are not considered as receptors and showing a mitochondrial localization possibly corresponding to monoamine oxidase [37,38], are removed from rat h eart membrane preparations. Figure 6 shows [ 3 H]clonidine displacement from I 1 -R present in rat heart membranes by N-amidino-2-hydroxypyrrolidine, agmatine, and cloni- dine. As observed in other target tissues [25], the specific binding of [ 3 H]clonidine to rat h eart membranes is s aturable (data not shown). Moreover, specific bindin g amounts to 3650 ± 294 d.p.m.Æh )1 Æ(mg p rotein) )1 , at saturating [ 3 H]clonidine concentration (¼ 1.0 · 10 )8 M ). N-amidino- 2-hydroxypyrrolidine and agmatine are more efficient than clonidine in displacing [ 3 H]clonidine from specific binding sites in heart rat membranes, values o f IC 50 being Fig. 5. N-Amidino-2-hydroxypyrrolidine binding mode tohuman NOS-II (top) and bovine trypsin (bottom). The conformations of N-amidino- 2-hydroxypyrrolidine in the enzyme:inhibitor complexes were ob tained after 10 ps molecular dynamics. For further details, see text. Fig. 6. Competition of N-amidino-2-hydroxypyrrolidine (circles), agmatine (triangles), and clonidine (squares) with [ 3 H]clonidine for its specific binding sites in rat heart membranes. The filled diamond indi- cates [ 3 H]clonidine saturating s pec ific binding ( a ¼ 1) in the a bsence of the ligand (i.e. clonidine, or agmatine or N-amidino-2-hydroxypyrro - lidine). The continuo us lines were calculated according to Eqn (3) with the following IC 50 values: N-am idino-2-hydroxypyrrolidine and agmatine, IC 50 ¼ (1.3 ± 0.4) · 10 )9 M , and clonidine, IC 50 ¼ (2.2 ± 0.4) · 10 )8 M . Data were obtained at pH 7.4 and 37.0 °C, mean ± SD. For further details, see text. 890 P. Ascenzi et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (1.3 ± 0.4) · 10 )9 M and (2.2 ± 0.4) · 10 )8 M , respec- tively (at pH 7.4 and 37.0 °C) (Fig. 6). DISCUSSION For the first time, N-amidino-2-hydroxypyrrolidine, the product of agmatine oxidation by P. sativum copper amine oxidase, has been identified an d characterized from the structural and biochemical viewpoints. Notably, the enzy- matic oxidation of agmatine leads to the cyclic compound N-amidino-2-hydroxypyrrolidine, as the only detectable reaction product (Figs 2 and 3). In f act, the formation of 4-guanidinobutyraldehyde was never observed. Therefore, 4-guanidinobutyraldehyde, the best substrate of the alde- hyde dehydrogenase that occurs in Fabaceae plants a nd rat hepatocytes with copper amine oxidase [39–42], does not appear to originate from the enzymatic cycling of agmatin e to N-amidino-2-hydroxypyrrolidine. N-Amidino-2-hydroxypyrrolidine inhibits competitively NOS-I, NOS-II, and trypsin (Fig. 4). This compound binds to the Glu597 and Glu371 carboxylate, present in NOS-I and NOS-II, respectively (Glu296 in human NOS- II; see Fig. 5), which is required for substrate ( i.e. L -arginine) recognition [21,33]. Moreover, N-amidino-2- hydroxypyrrolidine binds to the trypsin primary specificity subsite S 1 forming a salt bridge with the Asp 189 carboxylate (Fig. 5). The latter is required for recognition of the cationic amino acid residue present at the P 1 position of substrates and inhibitors of trypsin-like serine proteinases [35,36]. N-Amidino-2-hydroxypyrrolidine and agmatine displace efficiently [ 3 H]clonidine from I 1 -R present in heart rat membranes (Fig. 6). Interestingly, d ifferent physiological roles (i.e. neuronal neurotransmission and hypotensive protection of cardiovascular system) have been linked to agmatine, which has b een reported to be the endogenous ligand for I-R 1 [7] and to represent the N-amidino- 2-hydroxypyrrolidine precursor. In this respect, pleiotropic functional role(s) of N-amidino-2-hydroxypyrrolidine may be envisaged, as reported for agmatine [7]. As a w hole, agmatine oxidation by P. sativum copper amine oxidase may represent a new biocatalytic route for the synthesis of N-amidino-2-hydroxypyrrolidine, possibly representing a lead compound for the development of NOS and trypsin-like s erine p rotease i nhibitors. Moreover, N-amidino-2-hydroxypyrrolidine may represent a new ligand for I 1 -R. ACKNOWLEDGEMENTS Authors wish to t hank Prof S. Aim e and Dr G. Rea for helpful discussions and Dr L. Leone and Mr A. Merante for technical assistance. This study was partially supported by grants from the National Research Council of Italy (CNR, target oriented project ÔBiotechnologyÕ, 99.00280.PF49 to P. A., a nd 99.00360.PF49 to M . F.). Access to t he 6 00 MHz NMR facility h as be en g ranted b y Bioindustry Park Canavese, Colleretto Giacosa, TO, Italy. REFERENCES 1. McIntire, W.S. & Hartmann, C. 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