Agmatine oxidation by copper amine oxidasePaolo Ascenzi1,*, Mauro Fasano2,*, Maria Marino1, Giorgio Venturini1and Rodolfo Federico1 1 Department of Biology, University ÔRoma TreÕ, Rome,
Trang 1Agmatine oxidation by copper amine oxidase
Paolo Ascenzi1,*, Mauro Fasano2,*, Maria Marino1, Giorgio Venturini1and Rodolfo Federico1
1
Department of Biology, University ÔRoma TreÕ, Rome, Italy;2Department of Structural and Functional Biology,
University of Insubria, Varese, Italy
The product of agmatine oxidation catalyzed by Pisum
sativum L copper amine oxidase has been identified by
means of one- and two-dimensional1H-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 bovine trypsin
(trypsin) activity, values of Kibeing (1.1 ± 0.1)· 10)5M(at
pH 7.5 and 37.0°C), (2.1 ± 0.1) · 10)5M(at pH 7.5 and
37.0°C), and (8.9 ± 0.4) · 10)5M(at pH 6.8 and 21.0°C),
respectively Remarkably, the affinity of
N-amidino-2-hydroxypyrrolidine for NOS-I, NOS-II and trypsin is
significantly higher than that observed for agmatine and clonidine binding Furthermore, N-amidino-2-hydroxy-pyrrolidine and agmatine are more efficient than clonidine in displacing [3H]clonidine (¼ 1.0 · 10)8M) from specific binding sites in heart rat membranes, values of IC50being (1.3 ± 0.4)· 10)9M and (2.2 ± 0.4)· 10)8M, respec-tively (at pH 7.4 and 37.0°C)
Keywords: copper amine oxidase; agmatine; N-amidino-2-hydroxypyrrolidine; enzyme inhibition; type 1 imidazoline receptor binding
Copper amine oxidase has been identified in bacteria, yeasts,
fungi, plants, and animals This enzyme is a homodimer of
70- to 90-kDa subunits, each containing a single copper ion
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 preferences depending
upon the enzyme source [1–5] The copper amine oxidase
catalyzed reactions follow the general scheme:
Eox þ R-CH2-NH2! Ered þ R-CHO ðreaction 1Þ
Ered þ O2 þ H2O! Eox þ NH3 þ H2O2 ðreaction 2Þ
where Eoxrepresents the enzyme–quinone, R-CH2-NH2is
the substrate, Eredis 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, catalyzed 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, being identified as a novel neuro-transmitter and modulator of cardiovascular functions via binding to type 1 imidazoline (I1-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 on the role played by the product(s) of agmatine metabolism on cell function(s) Here, the biosynthesis and the biochemical characterization of N-amidino-2-hydroxypyrrolidine, the product of agmatine oxidation by Pisum sativum L copper amine oxidase, is reported
M A T E R I A L S A N D M E T H O D S
Proteins
P sativumcopper 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)2MHepes buffer), 5.0· 10)4M EGTA, 1.0· 10)3M 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 55176321, 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 2001, revised 17 October 2001, accepted 3 December
2001)
Trang 2NOS-I and NOS-II containing homogenates were desalted
by chromatography over disposable PD-10 columns packed
with Sephadex G-25 medium (Amersham Pharmacia
Bio-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 colilipopolysaccharide (serotype 0127:B8) were
obtained from Sigma Chemical Co [3H]L-arginine (specific
activity 2.0 TBqÆmmol)1) and [3H]clonidine (specific activity
2.6 TBqÆmmol)1) were purchased from NENTM Life
Science Products (Boston, MA, USA) Deuterium oxide
(99.8% isotopic enrichment) was obtained from Cortec
(Paris, France) All the other chemicals were from Merck
AG (Darmstadt, Germany) All products were of analytical
or reagent grade and used without further purification
Animals
Male Sprague–Dawley rats (from Morini, Italy), 4- to
5-month-old, were housed and acclimatized for 1 week under
controlled temperature (20 ± 1°C), humidity (55 ± 10%),
and light (from 7 a.m to 7 p.m) conditions The rats were
anaesthetized with ether in a fume hood, and organs
removed and rapidly chilled in liquid nitrogen (brain
and lung) or in ice-cold medium solution (2.0· 10)2M
NaHCO3; heart) Animal experiments were performed
accor-ding to ethical guidelines for the conduct of animal research
P sativum copper amine oxidase assay
Oxidation of agmatine by P sativum copper amine
oxi-dase was investigated spectrophotometrically by
follo-wing the formation of a pink adduct (e515nm¼ 2.6 ·
104M )1Æcm)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)1M
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)1M phosphate buffer, pH 7.0)
were added to a buffered solution (1.0 mL; 1.0· 10)1M
phosphate buffer, pH 7.0) containing the substrate (i.e
agmatine), aminoantipyrine (1.0· 10)4M),
3,5-dichloro-2-hydroxybenzenesulfonic acid (1.0· 10)3M), and
horse-radish peroxidase (1.5· 10)6M) 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 O2
consumption, at pH 7.0 (1.0· 10)1M phosphate buffer)
and 25.0°C [12] In a typical experiment, 20 lL of a
buffered agmatine solution (1.0· 10)1Mphosphate buffer,
pH 7.0) were added to a buffered solution (1.0 mL;
1.0· 10)1M phosphate buffer, pH 7.0) containing
P sativumcopper amine oxidase The initial velocity for
the enzymatic oxidation of agmatine was then measured
In the enzyme assay, the P sativum copper amine oxidase concentration was 5.0· 10)9Mand the agmatine concen-tration ranged between 5.0· 10)5Mand 5.0· 10)3M The enzyme activity was linear up to 5 min of incubation and results were expressed as lmol productÆs)1Æ(lmol enzyme))1 Under all the experimental conditions, the 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 equilibraincuba-tion time was very short, being completed within the mixing time ( 15 s)
Values of the first-order rate-limiting catalytic constant (kcat) and of the Michaelis constant, as determined in the absence of the inhibitor (K0m) for the P sativum copper amine oxidase catalyzed oxidation of agmatine, were obtained from the dependence of the initial velocity for agmatine oxidation (vi) on the substrate (i.e agmatine) concentration ([S]), according to Eqn (1) [13]:
vi ¼ kcat½S=ðK0
Values of kcat and K0
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)4M, respectively, at pH 7.0 (1.0·
10)1M phosphate buffer) and 25.0°C (Fig 1) Values of
kcatand K0
mare independent of the enzyme assay
Biosynthesis ofN-amidino-2-hydroxypyrrolidine N-Amidino-2-hydroxypyrrolidine was synthesized as fol-lows Twenty micrograms of P sativum copper amine oxidase were added to 1.0 mL of a buffered 2.0· 10)3M
agmatine solution (5.0· 10)2Mphosphate buffer, pH 7.4)
28 lg of bovine catalase were also added to the reaction solution (1.0 mL) in order to remove H2O2, arising from the
P sativum copper amine 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 PM10 membranes (Amicon, Inc., Beverly, MA, USA)
Fig 1 Effect of substrate (i.e agmatine) concentration on values of v i
for the P sativum copper amine oxidase catalyzed oxidation of agma-tine The continuous line was calculated according to Eqn (1), with the following values of k cat (¼ 1.3 ± 0.1 s)1) and K 0
m [¼ (3.8 ± 0.3) ·
10)4M ] Data were obtained at pH 7.0 and 25.0 °C, mean ± SD For further details, see text.
Trang 3The total conversion of agmatine to
N-amidino-2-hydroxypyrrolidine was detected by1H-NMR spectroscopy
Moreover, the agmatine/N-amidino-2-hydroxypyrrolidine
stoichiometry is 1 : 1 as shown by1H-NMR spectroscopy
The N-amidino-2-hydroxypyrrolidine concentration was
determined from 100% conversion of agmatine to
N-amidino-2-hydroxypyrrolidine as demonstrated by
1H-NMR spectroscopy
Under all the experimental conditions, the formation of
free 4-guanidinobutyraldehyde was observed neither by
the o-aminobenzaldehyde assay [14] (data not shown) nor
1H-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 spectrum was recorded prior to addition
of P sativum copper amine oxidase.1H-NMR one- and
two-dimensional spectra were recorded at 25.0°C on a
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 suppressed
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 of the agmatine oxidation
product was assigned by COSY, by setting the flip angle of
the second pulse to 35° To this purpose, 256 t1increments
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 theN-amidino-2-hydroxypyrrolidine structure
Energy minimization of the proposed structure of
N-amidino-2-hydroxypyrrolidine was 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 [3H]L-arginine to [3H]L-citrulline at pH 7.5
(5.0· 10)2MHepes 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 added
to the reaction mixture (100 lL) containing 1.0· 10)3M
NADPH, 1.2· 10)3M CaCl2, 1.0 lgÆmL)1 calmodulin, 1.0· 10)5M FAD, 1.0· 10)5M FMN, [3H]L-arginine (from 12 to 185 kBq) and L-arginine (from 1.0· 10)6M
to 1.0· 10)4M), in the absence and presence of N-ami-dino-2-hydroxypyrrolidine (from 5.0· 10)6M and 5.0·
10)5M) For the determination of NOS-II activity, CaCl2 and calmodulin were omitted, and 1.0· 10)3MEGTA was added to the reaction mixture NOS-I and NOS-II activity was assayed in the presence of 5.0· 10)5MBH4[16] In the enzyme assay, the NOS-I or NOS-II concentration was 2.0· 10)7M After 15 min incubation, the reaction was stopped by addition of an ice-cold 2.0· 10)2M Hepes buffer solution (700 lL), pH 5.5, containing 2.0· 10)3 M
EDTA [3H]L-citrulline was separated from [3H]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)1Æ(mg protein))1 Under all the experimental conditions, the initial velocity for NOS-I and NOS-II catalyzed conversion ofL-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 (kcat) and of the Michaelis constant, as determined in the absence and presence of the inhibitor (K0m and Kappm , respectively), for NOS-I and NOS-II catalyzed conversion
ofL-arginine toL-citrulline were obtained from the depen-dence of the initial velocity for substrate conversion (vi) on theL-arginine concentration ([S]), according to Eqn (1) [13] Values of kcatand K0
m for the NOS-I catalyzed conversion
of L-arginine to L-citrulline were 1.4 ± 102pmol prod-uctÆmin)1Æ(mg protein))1and 4.0· 10)6M, respectively, at
pH 7.5 and 37.0°C [11] Values of kcat and K0
m for the NOS-II catalyzed conversion ofL-arginine to L-citrulline were 4.7· 101pmol productÆmin)1Æ(mg protein))1 and 1.8· 10)5M, 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)6M), added
to the NOS-I and NOS-II preparations, to met-hemoglobin,
Fig 2.1H-NMR spectra of 2.0 · 10)3M
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 The residual water signal was suppressed
by presaturation For further details, see text.
Trang 4in the presence of N-amidino-2-hydroxypyrrolidine as the
substrate instead of L-arginine, at pH 7.5 (5.0· 10)2M
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)1M phosphate buffer) and
21.0°C [20], in the absence and presence of
N-amidino-2-hydroxypyrrolidine In a typical experiment, 20 lL of a buffered trypsin solution (1.0· 10)1M phosphate buffer,
pH 6.8) were added to 1.0 mL of a buffered solution (1.0· 10)1M 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-hydroxypyrrolidine) 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)6M, the N-a-benzoyl-L-arginine p-nitroanilide concentration ranged between 1.0· 10)5M and 1.0· 10)3M, and the N-ami-dino-2-hydroxypyrrolidine concentration ranged between 2.0· 10)5M and 8.0· 10)5M The enzyme activity was linear up to 10 min of incubation and results were expressed
as lmol productÆs)1Æ(lmol enzyme))1 Under all the exper-imental conditions, the initial velocity for the trypsin catalyzed hydrolysis of N-a-benzoyl-L-arginine p-nitroani-lide was unaffected by the enzyme/inhibitor/substrate incubation time In fact, 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 (kcat) and of the Michaelis constant determined in the absence and presence of the inhibitor (K0
m and Kapp
m , respectively) for the trypsin catalyzed hydrolysis of N-a-benzoyl-L-arginine p-nitroanilide were obtained from the dependence of the initial velocity for substrate hydrolysis (vi) on the N-a-benzoyl-L-arginine p-nitroani-lide concentration ([S]), according to Eqn (1) [13] Values
of kcat and K0m for the trypsin catalyzed hydrolysis of N-a-benzoyl-L-arginine p-nitroanilide were 0.70 s)1 and 3.0· 10)4M, respectively, at pH 6.8 and 21.0°C [20]
Determination of values of the inhibition dissociation equilibrium constant (Ki) forN-amidino-2-hydroxypyrrolidine binding
to NOS-I, NOS-II, and trypsin Values of the inhibition dissociation equilibrium constant (Ki) for the competitive inhibition of the NOS-I and NOS-II catalyzed conversion ofL-arginine toL-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 Kappm /K0mratio on the inhibitor concentration (i.e [I]), according to Eqn (2) [13]:
Kappm =K0m ¼ Kiÿ 1½I þ 1 ð2Þ
As expected for a simple competitive inhibition system [13], values of kcat for the NOS-I and NOS-II catalyzed conversion ofL-arginine toL-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, 16 dummy scans,
relaxation delay 2 s Labels refer to the resonance assignment in
Fig 1B For further details see text.
Trang 5isothiourea complex (PDB accession no 4NOS) [21] and the
bovine trypsin:benzamidine adduct (PDB accession no
1CE5) [22] as templates, respectively The atomic
coordi-nates of rat NOS-II are not yet available [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 Energy minimization and molecular
dynamics were performed on a Silicon Graphics O2
workstation (SGI, Irvine, CA, USA) withHYPERCHEM4.5
for SGI (Hypercube Inc., Gainesville, FL, USA)
I1-R binding assay
Cardiac muscle (cleaned of connective tissue and fat) was
finely minced and homogenized in ice-cold medium solution
2.0ở 10)2M NaHCO3, containing 1.0ở 10)4M
phen-ylmethanesulfonyl fluoride, with a wet weight to volume
ratio of 1 : 7, using a glass-Teflon homogenizer (10ở 30 s)
[24] The homogenate was centrifuged at 1500 g for 15 min
(4.0ồC) The supernatant was centrifuged at 45 000 g for
5 min (at 4.0ồC) The pellet was washed twice, then
re-suspended in 2 mL of ice-cold 5.0ở 10)3MHepes buffer,
containing 5.0ở 10)4MEGTA, 5.0ở 10)4MMgCl2, and
1.0ở 10)4M ascorbic acid (pH 7.4) [25] Membrane
pre-parations were free of mitochondria 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
[3H]clonidine at 37.0ồC in a final volume of 0.5 mL of
5.0ở 10)3MHepes buffer, containing 5.0ở 10)4MEGTA,
5.0ở 10)4M MgCl2, and 1.0ở 10)4M ascorbic acid
(pH 7.4) The reaction was stopped by rapid vacuum
filtration with a Millipore harvester through Whatman GF/C
glass fiber filters (Whatman International Ltd Maidstone,
UK) presoaked with 10% polyethyleneglycol in Tris/HCl
2.0ở 10)2M, containing MgCl21.0ở 10)2M, followed by
rapid washing of filters with 10 mL ice-cold 5.0ở 10)3M
Hepes buffer, containing 5.0ở 10)4MEGTA, 5.0ở 10)4M
MgCl2, and 1.0ở 10)4M ascorbic acid (pH 7.4) Filters
were placed in a 6-mL scintillation fluid and the
radio-activity determined by liquid scintillation counting
Epine-phrine (1.0ở 10)5M), which does not bind to imidazoline
sites [26,27], was added to the assay to prevent [3H]clonidine
from binding to a-adrenergic receptors Nonspecific binding
was defined as [3H]clonidine-binding (the [3H]clonidine
concentration ranged between 1.5ở 10)4M and
5.0ở 10)4M) Saturation studies were performed with
1.0ở 10)8M [3H]clonidine and increasing concentrations
of the unlabelled ligand (i.e
N-amidino-2-hydroxypyrroli-dine, agmatine, and clonidine; from 1.0ở 10)9M to
1.0ở 10)6M) Protein concentration was measured by the
method of Bradford [28], using bovine serum albumin as the
standard
Values of IC50for [3H]clonidine displacement from I1-R
in heart rat membranes by
N-amidino-2-hydroxypyrroli-dine, agmatine, and clonidine were determined according to
Eqn (3):
a Ử 1=f1 ợ đơL=IC50ỡg đ3ỡ
where a is the molar fraction of [3H]clonidine bound to I1-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]
R E S U L T S
Over the whole substrate (i.e agmatine) concentration range explored (i.e between 5.0ở 10)5M and 5.0ở 10)3M), the P sativum copper amine oxidase cata-lyzed oxidation of agmatine follows simple MichaelisỜ Menten kinetics (Fig 1) According to the literature [30], values of kcat and K0m for the P sativum copper amine oxidase catalyzed oxidation of agmatine are 1.3 ổ 0.1 s)1 and (3.8 ổ 0.3)ở 10)4M, respectively, at pH 7.0 and 25.0ồC Moreover, values of kcatand K0mwere independent
of the enzymatic assay used (spectrophotometric vs pola-rographic) The stoichiometric analysis of the enzymatic oxidation of agmatine yields a molar ratio of substrate (i.e agmatine) to O2and H2O2of 1 : 1 : 1
Figure 2 shows the 1H-NMR spectra of agmatine before (Fig 2A) and after (Fig 2B) oxidation catalyzed
by P sativum copper amine oxidase, at pD 7.4 and 25.0ồC The agmatine sample shows some signals at the 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 Ử 5.5 p.p.m., and (b) the splitting of CH2 signals in magnetically unequivalent components On the basis of the general mechanism (see reactions 1 and 2), one triplet (relative area 1) should occur at about dỬ 9 p.p.m., corresponding to the formyl proton, one triplet at about dỬ 3 p.p.m (relative area 2), and two multiplets at about dỬ 2 p.p.m (relative area 2 each) As the -CHO signal was not observed, the formation of the corresponding free aldehyde (i.e 4-guanidobutyraldehyde) was ruled out To note that the agmatine/N-amidino-2-hydroxypyrrolidine stoichiometry
is 1 : 1 as shown by1H-NMR spectroscopy
A possible explanation for the resolution of the magnetic equivalence of CH2 groups would be the formation of an intramolecular Schiff base in its emiac-etalic form, deriving from nucleophilic attack of the guanidinic eN nitrogen to the (transient) aldehydic carbonyl This implies the formation of a chiral center
on the ring, with all CH2protons 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 occur 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 sativumcopper 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 of3J scalar couplings [15] As three-bond couplings were not observed, it was assumed that the involved protons form dihedral angles close to 90ồ [31] In other words, the absence
of scalar coupling between A and, 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 oxidation catalyzed by P sativum copper amine oxidase) after 200 cycles of energy
Trang 6minimi-zation in the MMFF force field [32], with torsion angles
constrained according to the results of the COSY spectrum
(Fig 3, top panel)
As shown in Fig 4, N-amidino-2-hydroxypyrrolidine inhibits competitively the 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 Ki 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 binding (see Table 1) As reported for agmatine [8] and 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 the sub-strate instead ofL-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 concentration (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) catalyzed hydrolysis of N-a-benzoyl- L -arginine p-nitroanilide The continuous lines were cal-culated according to Eqn (2) with values of K i given in Table 1 Data were obtained between pH 6.8 and 7.5 and between 21.0 °C and 37.0 °C, mean ± SD, for further details, see 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)5 a (6.6 ± 1.1) · 10)4 b (5.0 ± 0.2) · 10)3 c NOS-II (2.1 ± 0.1) · 10)5 a (2.2 ± 0.2) · 10)4 b >5 · 10)2 c
Trypsin (8.9 ± 0.4) · 10)5 d >10)2 e >10)2 e
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].
Trang 7lidine complexes In human NOS-II (top panel),
N-amidino-2-hydroxypyrrolidine is hosted in the hydrophobic cavity
defined by the heme prosthetic 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 forL-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 specificity subsite S1 (bottom panel) Interestingly, the alicyclic group is extended in a semichair conformation, with the positively charged amidino group of N-amidino-2-hydroxypyrrolidine 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 the P1position of substrates and inhibitors of trypsin-like serine proteinases [35,36] N-Amidino-2-hydroxypyrrolidine, agmatine, and cloni-dine bind to I1-binding sites (i.e I1-R) In fact, the I2sites, which are not considered as receptors and showing a mitochondrial localization possibly corresponding to monoamine oxidase [37,38], are removed from rat heart membrane preparations Figure 6 shows [3H]clonidine displacement from I1-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 [3H]clonidine to rat heart membranes is saturable (data not shown) Moreover, specific binding amounts to
3650 ± 294 d.p.m.Æh)1Æ(mg protein))1, at saturating [3H]clonidine concentration (¼ 1.0 · 10)8M) N-amidino-2-hydroxypyrrolidine and agmatine are more efficient than clonidine in displacing [3H]clonidine from specific binding sites in heart rat membranes, values of IC being
Fig 5 N-Amidino-2-hydroxypyrrolidine binding mode to human NOS-II
(top) and bovine trypsin (bottom) The conformations of
N-amidino-2-hydroxypyrrolidine in the enzyme:inhibitor complexes were obtained
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 [3H]clonidine for its specific binding sites in rat heart membranes The filled diamond indi-cates [ 3 H]clonidine saturating specific binding (a ¼ 1) in the absence of the ligand (i.e clonidine, or agmatine or N-amidino-2-hydroxypyrro-lidine) The continuous lines were calculated according to Eqn (3) with the following IC 50 values: N-amidino-2-hydroxypyrrolidine and agmatine, IC 50 ¼ (1.3 ± 0.4) · 10)9M , and clonidine, IC 50 ¼ (2.2 ± 0.4) · 10)8M Data were obtained at pH 7.4 and 37.0 °C, mean ± SD For further details, see text.
Trang 8(1.3 ± 0.4)· 10)9M and (2.2 ± 0.4)· 10)8M,
respec-tively (at pH 7.4 and 37.0°C) (Fig 6)
D I S C U S S I O N
For the first time, N-amidino-2-hydroxypyrrolidine, the
product of agmatine oxidation by P sativum copper amine
oxidase, has been identified and 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 fact, the formation of
4-guanidinobutyraldehyde was never observed Therefore,
4-guanidinobutyraldehyde, the best substrate of the
alde-hyde dehydrogenase that occurs in Fabaceae plants and rat
hepatocytes with copper amine oxidase [39–42], does not
appear to originate from the enzymatic cycling of agmatine
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
I and 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 S1 forming a salt bridge with the Asp189
carboxylate (Fig 5) The latter is required for recognition
of the cationic amino acid residue present at the P1
position of substrates and inhibitors of trypsin-like serine
proteinases [35,36]
N-Amidino-2-hydroxypyrrolidine and agmatine displace
efficiently [3H]clonidine from I1-R present in heart rat
membranes (Fig 6) Interestingly, different physiological
roles (i.e neuronal neurotransmission and hypotensive
protection of cardiovascular system) have been linked to
agmatine, which has been reported to be the endogenous
ligand for I-R1 [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 whole, 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 serine protease inhibitors Moreover,
N-amidino-2-hydroxypyrrolidine may represent a new
ligand for I1-R
A C K N O W L E D G E M E N T S
Authors wish to thank Prof S Aime 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., and 99.00360.PF49 to M F.).
Access to the 600 MHz NMR facility has been granted by Bioindustry
Park Canavese, Colleretto Giacosa, TO, Italy.
R E F E R E N C E S
1 McIntire, W.S & Hartmann, C (1993) Copper-containing amine
oxidases In Principles and Applications of Quinoproteins (Davison,
V.L., ed.), pp 99–171 Marcel Dekker, New York.
2 Fontecave, M & Eklund, H (1995) Copper amine oxidase: a novel use for a tyrosine Structure 3, 1127–1129.
3 Klinman, J.P (1996) Mechanisms whereby mononuclear copper proteins functionalize organic substrates Chem Rev 96, 2541– 2561.
4 Buffoni, F & Ignesti, G (2000) The copper-containing amine oxidases: biochemical aspects and functional role Mol Genet Metab 71, 559–564.
5 Federico, R., Angelini, R., Ercolini, L., Venturini, G., Mattevi, A.
& Ascenzi, P (1997) Competitive inhibition of swine kidney copper amine oxidase by drugs: amiloride, clonidine, and gabexate mesylate Biochem Biophys Res Commun 240, 150–152.
6 Holt, A & Baker, G.B (1995) Metabolism of agmatine (clonidine-displacing substance) by diamine oxidase and the possible impli-cations for studies of imidazoline receptors Prog Brain Res 106, 187–197.
7 Reis, D.J & Regunathan, S (2000) Is agmatine a novel neuro-transmitter in brain? Trends Pharmacol Sci 21, 187–193.
8 Galea, E., Regunathan, S., Eliopoulos, V., Feinstein, D.L & Reis, D.J (1996) Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation
of arginine Biochem J 316, 247–249.
9 Demady, D.R., Jianmongkol, S., Vuletich, J.L., Bender, A.T & Osawa, Y (2001) Agmatine enhances the NADPH oxidase activity of neuronal NO synthase and leads to oxidative inacti-vation of the enzyme Mol Pharmacol 59, 24–29.
10 McGuirl, M.A., McCahon, C.D., McKeown, K.A & Dooley, D.M (1994) Purification and characterization of pea seedling amine oxidase for crystallization studies Plant Physiol 106, 1205– 1211.
11 Stuehr, D.J & Griffith, O.W (1996) Purification, assay and properties of mammalian nitric oxide synthases In Methods in Nitric Oxide Research (Feelisch, M & Stamler, J.S., eds), pp 177–
186 John Wiley & Sons Ltd, Chichester.
12 Rinaldi, A., Floris, G & Finazzi-Agro`, A (1982) Purification and properties of diamine oxidase from Euphorbia latex Eur J Biochem 127, 417–422.
13 Ascenzi, P., Ascenzi, M.G & Amiconi, G (1987) Enzyme competitive inhibition: graphical determination of K i and presen-tation of data in comparative studies Biochem Educ 15, 134– 135.
14 Holmestedt, B., Larsson, L & Tham, R (1961) Further studies
on spectrophotometric method for the determination of amine oxidase activity Biochim Biophys Acta 48, 182–186.
15 Braun, S., Kalinowski, H.-O & Berger, S (1998) 150 and More Basic NMR Experiments Wiley-VCH, Weinheim.
16 Venturini, M., Colasanti, M., Persichini, T., Fioravanti, E., Federico, R & Ascenzi, P (2000) Selective inhibition of nitric oxide synthase type I by clonidine, an antihypertensive drug Biochem Pharmacol 60, 539–544.
17 Venturini, G., Colasanti, M., Fioravanti, E., Bianchini, A & Ascenzi, P (1999) Direct effect of temperature on the catalytic activity of nitric oxide synthases types I, II, and III Nitric Oxide 3, 375–382.
18 Feelisch, M., Kubitzek, D & Werringloer, J (1996) The oxyhe-moglobin assay In Methods in Nitric Oxide Research (Feelish, M.
& Stamler, J.S., eds), pp 455–478 John Wiley & Sons Ltd, Chichester.
19 Venturini, G., Menegatti, E & Ascenzi, P (1997) Competitive inhibition of nitric oxide synthase by p-aminobenzamidine, a serine proteinase inhibitor Biochem Biophys Res Commun 232, 88–90.
20 Ascenzi, P., Menegatti, E., Guarneri, M., Bortolotti, F & Anto-nini, E (1982) Catalytic properties of serine proteases 2 Com-parison between human urinary kallikrein and human urokinase, bovine b-trypsin, bovine thrombin, and bovine a-chymotrypsin Biochemistry 21, 2483–2490.
Trang 921 Fischmann, T.O., Hruza, A., Niu, X.D., Fossetta, J.D., Lunn,
C.A., Dolphin, E., Prongay, A.J., Reichert, P., Lundell, D.J.,
Narula, S.K & Weber, P.C (1999) Structural characterization of
nitric oxide synthase isoforms reveals striking active-site
conser-vation Nat Struct Biol 6, 233–242.
22 Ota, N., Stroupe, C., Ferreira da Silva, J.M.S., Shah, S.S.,
Mares-Guia, M & Brunger, A.T (1999) Non-Boltzmann
Thermo-dynamic Integration (NBTI) for macromolecular systems: relative
free energy of binding of trypsin to benzamidine and benzylamine.
Proteins 37, 641–653.
23 Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N.,
Weissig, H., Shindyalov, I.N & Bourne, P.E (2000) The Protein
Data Bank Nucleic Acids Res 28, 235–242.
24 Glossmann, H & Ferry, D.R (1985) Assay for calcium channels.
Methods Enzymol 109, 513–550.
25 Molderings, G.J., Donecker, K & Go¨thert, M (1995)
Charac-terization of non-adrenergic [ 3 H]clonidine binding sites in rat
stomach: high affinity of imidazolines, guanidines and sigma
ligands Naunyn-Schmiedeberg’s Arch Pharmacol 351, 561–564.
26 Molderings, G.J., Moura, D., Fink, K., Bo¨nisch, H & Go¨thert,
M (1993) Binding of [3H]clonidine to I 1 -imidazoline sites in
bovine adrenal medullary membranes Naunyn-Schmiedeberg’s
Arch Pharmacol 348, 70–76.
27 Molderings, G.J., Kundt, L & Go¨thert, M (1994) [3H]Idazoxan
binding to bovine adrenal medullary membranes: identification
and pharmacological characterization of I 2 -imidazoline sites.
Naunyn-Schmiedeberg’s Arch Pharmacol 350, 252–257.
28 Bradford, M.M (1976) A rapid, sensitive method for the
quanti-fication of mg quantities of protein, utilizing the principle of
protein-dye binding Anal Biochem 72, 248–254.
29 Ascenzi, P., Desideri, A., Amiconi, G., Bertollini, A., Bolognesi,
M., Castagnola, M., Coletta, M & Brunori, M (1988) Effect of
inositol hexakisphosphate on the spectroscopic properties of the
nitric oxide derivative of ferrous naturally glycated human
hemoglobin HbA 1c J Inorg Biochem 34, 19–24.
30 Federico, R., Leone, L., Botta, M., Binda, C., Angelini, R.,
Venturini, G & Ascenzi, P (2001) Inhibition of pig liver and Zea
mays L polyamine oxidase: a comparative study J Enzyme Inhib.
16, 147–155.
31 Friebolin, H (1993) Basic One- and Two-Dimensional NMR Spectroscopy VCH, Weinheim.
32 Halgren, T.A (1996) Merck Molecular Force Field I Basis, form, scope, parametrization, and performance of MMFF94 J Com-put Chem 17, 490–519.
33 Crane, B.R., Arvai, A.S., Gachhui, R., Wu, C., Ghosh, D.K., Getzoff, E.D., Stuehr, D.J & Tainer, J.A (1997) The structure of nitric oxide synthase oxygenase domain and inhibitor complexes Science 278, 425–431.
34 Bode, W & Schwager, P (1975) The refined crystal structure of bovine b-trypsin at 1.8 A˚ resolution II Crystallographic refine-ment, calcium binding site, benzamidine binding site and active site at pH 7.0 J Mol Biol 98, 693–717.
35 Bode, W & Huber, R (1992) Natural protein proteinase inhibi-tors and their interaction with proteinases Eur J Biochem 204, 433–451.
36 Bode, W & Huber, R (2000) Structural basis of the endopro-teinase–protein inhibitor interaction Biochim Biophys Acta 1477, 241–252.
37 Tesson, F., Limon-Boulez, I., Urban, P., Puype, M., Van-dekerckhove, J., Coupry, I., Pompon, D & Parini, A (1995) Localization of I 2 -imidazoline binding sites on monoamine oxi-dases J Biol Chem 270, 9856–9861.
38 Ernsberger, P & Haxhiu, M.A (1997) The I 1 -imidazoline-binding site is a functional receptor mediating vasodepression via the ventral medulla Am J Physiol 273, R1572–R1579.
39 Matsuda, H & Suzuki, Y (1981) Purification and properties of the diamine oxidase from Vicia faba leaves Plant Cell Physiol 22, 737–746.
40 Matsuda, H & Suzuki, Y (1984) c-Guanidinobutyraldehyde dehydrogenase of Vicia faba leaves Plant Physiol 76, 654–657.
41 Kaneoke, M., Shimizu, E & Yorifuji, T (1994) Metabolism of L-arginine, agmatine, and related-compounds in Nocardioides simplex Biosci Biotechn Biochem 58, 244–249.
42 Cabella, C., Gardini, G., Corpillo, D., Testore, G., Bedino, S., Solinas, S.P., Cravanzola, C., Vargiu, C., Grillo, M.A & Colombatto, S (2001) Transport and metabolism of agmatine in rat hepatocyte cultures Eur J Biochem 268, 940–947.