Eur. J. Biochem. 1–13 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2004.04434.x
1,5-Diamino-2-pentyne is both a substrate and inactivator of plant
copper amine oxidases
Zbyněk Lamplot1, Marek Šebela1, Michal Maloň2, René Lenobel2, Karel Lemr3, Jan Havliš4, Pavel Peč1,
Chunhua Qiao5 and Lawrence M. Sayre5
1
Department of Biochemistry, 2Laboratory of Growth Regulators, and 3Department of Analytical Chemistry, Faculty of Science,
Palacký University, Olomouc, Czech Republic; 4Department of Analytical Chemistry, Faculty of Science, Masaryk University, Brno,
Czech Republic; 5Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA
1,5-Diamino-2-pentyne (DAPY) was found to be a weak
substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin
(Onobrychis viciifolia, OVAO) amine oxidases. Prolonged
incubations, however, resulted in irreversible inhibition of
both enzymes. For GPAO and OVAO, rates of inactivation
of 0.1–0.3 min)1 were determined, the apparent KI values
(half-maximal inactivation) were of the order of 10)5 M.
DAPY was found to be a mechanism-based inhibitor of the
enzymes because the substrate cadaverine significantly prevented irreversible inhibition. The N1-methyl and N5-methyl
analogs of DAPY were tested with GPAO and were weaker
inactivators (especially the N5-methyl) than DAPY. Prolonged incubations of GPAO or OVAO with DAPY resulted in the appearance of a yellow–brown chromophore
(kmax ¼ 310–325 nm depending on the working buffer).
Excitation at 310 nm was associated with emitted fluorescence with a maximum at 445 nm, suggestive of extended
conjugation. After dialysis, the color intensity was substantially decreased, indicating the formation of a low molecular
mass secondary product of turnover. The compound provided positive reactions with ninhydrin, 2-aminobenzaldehyde and Kovacs’ reagents, suggesting the presence of an
amino group and a nitrogen-containing heterocyclic structure. The secondary product was separated chromatographically and was found not to irreversibly inhibit GPAO.
MS indicated an exact molecular mass (177.14 Da) and
molecular formula (C10H15N3). Electrospray ionization- and
MALDI-MS/MS analyses yielded fragment mass patterns
consistent with the structure of a dihydropyridine derivative
of DAPY. Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2pentyne was identified by means of 1H- and 13C-NMR
experiments. This structure suggests a lysine modification
chemistry that could be responsible for the observed inactivation.
Copper-containing amine oxidases (CAOs, EC 1.4.3.6) play
a crucial role in the metabolism of primary amines. These
enzymes are widely distributed in nature [1]. In micro-
organisms, CAOs have a nutritional role in the utilization of
primary amines as the sole nitrogen and carbon source. In
mammals and plants, CAOs appear to be tissue specific, and
are implicated in wound healing, detoxification, cell growth,
signaling and apoptosis [1]. The oxidative deamination of
amine substrates catalyzed by CAOs yields the corresponding aldehydes with the concomitant production of hydrogen
peroxide and ammonia [2].
The reaction proceeds through a transamination mechanism mediated by an active site cofactor topaquinone. The
cofactor is derived from the post-translational self-processing of a specific tyrosine residue that requires both active site
copper and molecular oxygen [2]. The key step in the
oxidative deamination is conversion of the initial substrate
Schiff base (quinoimine) to a product Schiff base (quinoaldimine) facilitated by Ca proton abstraction via a
conserved aspartate residue acting as a general base at the
active site [3]. This step is followed by hydrolytic release of
the aldehyde product and the reduced cofactor is finally
reoxidized by molecular oxygen with the release of H2O2
and NH4+. The reduced topaquinone exists in two forms.
The first is an aminoresorcinol derivative coexisting with
Cu(II), which is in equilibrium with the second form, Cu(I)semiquinolamine radical [3]. The role of copper in the
reoxidation step has not been sufficiently elucidated for
Correspondence to M. Šebela, Department of Biochemistry, Faculty of
Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc,
Czech Republic. Fax: + 420 5856 34933; Tel.: + 420 5856 34927;
E-mail: [email protected]
Abbreviations: ABA, 2-aminobenzaldehyde; ACA, 6-aminocaproic
acid; BEA, 2-bromoethylamine; CAO, copper-containing amine
oxidase; DABY, 1,4-diamino-2-butyne; DAPY, 1,5-diamino-2-pentyne; DDD, 3,5-diacetyl-2,6-dimethyl-1,4-dihydropyridine; DMAB,
4-(dimethylamino)benzaldehyde; DMAC, 4-(dimethylamino)cinnamaldehyde; ESI, electrospray ionization; GPAO, grass pea (Lathyrus
sativus) amine oxidase; HABA, 2-(4-hydroxyphenylazo)benzoic acid;
IT, ion trap; LSAO, lentil (Lens esculenta) amine oxidase; MALDI,
matrix-assisted laser desorption/ionization; OVAO, sainfoin
(Onobrychis viciifolia) amine oxidase; PSAO, pea (Pisum sativum)
seedling amine oxidase; PSD, post source decay; Q, quadrupole;
TNBS, 2,4,6-trinitrobenzenesulfonic acid.
Enzyme: copper-containing amine oxidase (EC 1.4.3.6).
Note: A website is available at http://prfholnt.upol.cz/biochhp
(Received 24 August 2004, revised 23 September 2004,
accepted 13 October 2004)
Keywords: amine oxidase; diamine; mechanism-based inhibition; nuclear magnetic resonance; oxidation.
FEBS 2004
2 Z. Lamplot et al. (Eur. J. Biochem.)
most of the CAOs. For the amine oxidase from Hansenula
polymorpha, it seems likely that cofactor reoxidation
involves electron transfer from substrate-reduced topaquinone to oxygen that is bound at a site separate from
copper [2].
Plant CAOs prefer diamine substrates like putrescine and
cadaverine, hence they are also called diamine oxidases [4].
Inhibitors of these enzymes have recently been reviewed [5].
Among them, a special place is reserved for mechanismbased inhibitors, which undergo turnover-dependent conversion to electrophilic products capable of covalent binding
to an active-site nucleophile resulting in inactivation. Two
reported strategies in designing such inhibitors are the
incorporation of either halogen or unsaturation at the
b-position of amine substrates, examples being 2-bromoethylamine (BEA) [6] and 1,4-diamino-2-butyne (DABY)
[7], respectively. Namely, the inactivating effect of DABY
(as a putrescine analog) on plant CAOs has been studied in
detail at the molecular level [8–10]. Various b-unsaturated
compounds were tested in the reaction with bovine plasma
CAO [10–13]. Propargylic and chloroallylic diamines were
highly potent inhibitors of the enzyme, more so than simple
allylic diamines [11–13]. A recent study showed that the
homopropargyl amine, 1-amino-3-butyne, is also a potent
inactivator of certain CAOs [14]. For this reason, and
because it is an analog of cadaverine (pentane-1,5-diamine),
the best known substrate of plant CAOs [4], it seemed
important to determine the potential inactivating properties
of the higher DABY homolog, 1,5-diamino-2-pentyne
(DAPY). The unsymmetrical DAPY comprises both a
propargyl and homopropargyl amine.
DAPY was synthesized and tested as a substrate of two
plant CAOs. DAPY acts as a mechanism-based inhibitor of
the enzymes, causing their modification with the concomitant inactivation. However, in comparison with the effect of
DABY previously published [8], the modification extent is
considerably decreased. Only a few amino acid side chains
seem to be modified as a result of the reaction. A major part
of DAPY oxidation product, aminopentynal, after the
conjugate addition of an unreacted DAPY molecule, is
converted to a free nitrogenous heterocyclic compound,
whose dihydropyridine-derived structure was determined
using various analytical methods.
Materials and methods
Chemicals
The previously unreported DAPY dihydrochloride was
synthesized from the known 1,5-dichloro-2-pentyne [15] by
displacement of the activated propargylic chloride with
methanolic ammonia in a pressure bottle [11], tert-butoxycarbonyl protection of the introduced primary amine group,
displacement of the less reactive homopropargylic chloride
with methanolic ammonia in a pressure bottle (accompanied by an elimination side reaction), and finally HClmediated deprotection. Elemental analysis showed 33.38%
C, 7.41% H and 14.01% N (calculated 33.01, 7.08,
and 13.18%, respectively). Melting point: 177–179 C;
13
C-NMR spectrum (deuterium oxide): d 17.0, 29.4, 38.0,
74.3 and 82.8 p.p.m.; electrospray ionization ion trap mass
spectrometry (ESI-IT-MS) and MS/MS: a single quasimo-
lecular peak of m/z 99.1 providing fragment peaks of m/z
82.0 and 70.0. N5-Methyl-1,5-diamino-2-pentyne dihydrochloride was prepared as for DAPY, substituting methanolic methylamine in the penultimate step. N1-Methyl-1,5diamino-2-pentyne dihydrochloride was prepared by reaction of 1,5-dichloro-2-pentyne with aqueous methylamine,
and then with methanolic ammonia. Synthetic details and
characterization of these analogs are given elsewhere [16].
3,5-Diacetyl-2,6-dimethyl-1,4-dihydropyridine (DDD)
was prepared using the Hantzsch synthesis [17]. 2-Aminobenzaldehyde (ABA), bicinchoninic acid solution (Cat. No.
B9643), 4-(dimethylamino)benzaldehyde (DMAB), 4-(dimethylamino)cinnamaldehyde (DMAC), 3-hydroxypyridine, pyrrole and 2,4,6-trinitrobenzenesulfonic acid
(TNBS) solution (5%, w/v) were from Sigma (St. Louis,
MO, USA). Deuterium oxide (D2O, 99.96%) and d4methanol (CD3OD, 99.95%) were from Aldrich (Milwaukee, WI, USA). 6-Aminocaproic acid (ACA) and NADH
were supplied by Fluka (Buchs, Switzerland). 2-(4-Hydroxyphenylazo)benzoic acid (HABA) was from Bruker
Daltonik GmbH (Bremen, Germany). All other chemicals
were of analytical purity grade.
Enzymes
Plant diamine oxidases from grass pea (Lathyrus sativus,
GPAO) and sainfoin (Onobrychis viciifolia, OVAO) seedlings were prepared in homogeneous forms following
published protocols [18,19]. Specific activities assayed with
cadaverine as a substrate were 50 and 120 UÆmg)1, respectively. Bovine liver catalase (2000 UÆmg)1) and horseradish
peroxidase (100 UÆmg)1) were commercial products from
Fluka. Protein content in enzyme samples was estimated
using a standard method with bicinchoninic acid [20].
Kinetic measurements
CAO assay was carried out following a previously published
protocol [9]. The guaiacol spectrophotometric method was
used, which is based on a coupled reaction of horseradish
peroxidase [21]. Kinetic parameters of time-dependent
inactivation of the enzymes by DAPY were evaluated
according to the literature on mechanism-based inhibition
[11,22]. Various 0.1 M potassium phosphate buffers in the pH
range 5.0–8.0 were used in experiments performed to describe
the influence of pH on the inhibition potency of DAPY. To
assess the influence of ionic strength on the reaction, 0.1 M
Britton–Robinson buffer, pH 7.2, containing variable
potassium chloride was used. Rapid scanning of absorption
spectra of GPAO or OVAO mixed with DAPY under
admission of air was carried out by means of a DU-4500
spectrophotometer (Beckman, Fullerton, CA, USA) essentially as described previously [9]. Aerobic scans at longer time
intervals (10–120 min) after mixing GPAO or OVAO with
DAPY (1 : 100) were carried out using a Lambda 11
spectrophotometer (Perkin–Elmer, Ueberlingen, Germany).
TLC of DAPY oxidation product
TLC of the GPAO-DAPY reaction mixture was carried out
using commercial TLC plastic sheets (4 · 8 cm) with a
layer of Silica gel 60 F254 (Merck, Darmstadt, Germany);
FEBS 2004
n-propanol/MeOH/saturated sodium acetate solution
(40 : 3 : 60 v/v/v) was used as a mobile phase. Primary,
secondary and tertiary amino groups were detected using
ninhydrin, sodium nitroprusside and Dragendorff’s reagents, respectively. Aldehyde groups were detected using
Schiff’s reagent.
Spectrofluorimetry
A solution of DAPY (5 mM) in 20 mM potassium phosphate buffer, pH 7.0, was oxidized by an excess of GPAO at
23 C for 12 h. After that, the reaction mixture was filtered
using a centrifugal cartridge Microcon (Millipore, Bedford,
MA, USA), 0.5 mL, equipped with a 10 kDa cut-off filter.
The filtrate was used for spectrofluorimetry. Fluorescence
emission spectra of the DAPY oxidation product and
model compounds (DDD, 3-hydroxypyridine, NADH and
pyrrole) were obtained by means of an LS50B spectrofluorimeter (Perkin–Elmer, Boston, MA, USA). The oxidized
DAPY was measured with a fixed excitation at 310 nm.
Similarly, for the model compounds, the respective wavelengths of maximal absorption were taken as excitation
wavelengths.
Colorimetric trapping of DAPY oxidation product
For the various methods listed, absorption spectra were
recorded on Lambda 11 spectrophotometer against a blank
without DAPY. (a) Reaction with ABA [23]: DAPY
(2.5 mM) was oxidized by an excess of GPAO (500 nkat)
in 0.1 M potassium phosphate buffer, pH 7.0, in the presence
of catalase (200 U) and 2.5 mM ABA. After 1 h of
incubation at 30 C, 1 mL of 15% trichloroacetic acid was
added to the reaction mixture of the total volume 5 mL; (b)
Reaction with ninhydrin [24]: an aliquot (1 mL) of GPAO/
DAPY mixture (5 lM GPAO, 4 mM DAPY; initial concentrations) in 0.1 M potassium phosphate buffer, pH 7.0,
was taken out after 2 h of incubation at 23 C and mixed
with the same volume of warm ninhydrin reagent [24]. This
was followed by the addition of acetic acid (1.5 mL). The
sample was kept in a boiling water bath for 30 min to
develop the color. It was then cooled and 2.5 mL of acetic
acid was added to make up the volume to 6 mL. (c) Reaction with Kovacs’ reagent: an aliquot (1 mL) of GPAO/
DAPY mixture (2 lM GPAO, 0.2 mM DAPY; initial
concentrations) in 0.1 M potassium phosphate buffer,
pH 7.0, was removed after 90 min of incubation at 23 C
and mixed with 2 mL of the original Kovacs’ reagent
containing DMAB [7,8,25] (or its alternative contaning
DMAC [8]), incubated at 50 C for 30 min and cooled on ice
bath. In an alternative experiment, the GPAO/DAPY
reaction mixture was first separated by ultrafiltration using
the Microcon centrifugal cartridge as described above and
only the ultrafiltrate mixed with Kovacs’ reagent. Three
model compounds (DDD, pyrrole and NADH) were used to
compare spectral properties of their DMAB-adducts with
that of the DAPY oxidation product.
MS of DAPY reaction mixture
Samples for ESI-IT-MS were prepared by the oxidation of
5 mM buffered DAPY solution with an excess of GPAO.
DAPY inactivates plant amine oxidases (Eur. J. Biochem.) 3
Two buffers were used to optimize results: 0.1 M ammonium bicarbonate, pH 7.8, and 0.1 M Bistris/HCl, pH 7.0.
To evaluate the reactivity of the initial product aminoaldehyde, the reaction was also carried out in the presence of
5 mM ACA as a trapping nucleophilic reagent. After
incubation at 30 C for a sufficiently long time interval
(6–24 h), the reaction mixtures were separated by ultrafiltration using the Microcon centrifugal cartridge as given
above. The filtrate was properly diluted using methanol
before measurements. Mass spectra were obtained using an
ion trap mass spectrometer Finnigan MAT LCQ (Thermo
Electron Corp., San Jose, CA, USA) equipped with an ESI
interface. All samples were directly introduced to the
electrospray interface of the mass spectrometer by a syringe
at a flow rate of 5 lLÆmin)1. The ionization mode used
produced positively charged quasimolecular ions [M+H]+.
Parameters of the electrospray were as follows: source
voltage 5.6 kV, sheath gas flow 20 units, cone voltage
33.43 V, capillary temperature 250 C.
Enzymatic microscale production of DAPY oxidation
product
The larger quantity of DAPY oxidation product required
for further characterization was prepared by a cyclic flux
of DAPY solution through a hydroxyapatite column
(1 · 10 cm) containing immobilized GPAO and catalase.
After GPAO (10 mkat) and catalase (10 mkat) were loaded
in 10 mM ammonium bicarbonate, pH 7.8, the column was
washed with the same buffer. Then 50 mL of 5 mM DAPY
in 10 mM ammonium bicarbonate was left to circulate
through the column at 21 C using a peristaltic pump at a
flow rate of 1 mLÆmin)1 for 24 h. After stopping the cyclic
flux, the column was additionally washed with 20 mL of
10 mM ammonium bicarbonate, and the eluate was added
to the solution of oxidized DAPY. The combined solution
was then filtered using an ultrafiltration cell (100 mL)
equipped with a 10 kDa cut-off filter (Amicon, Danvers,
MA, USA). Water was removed on a rotary vacuum
evaporator Rotavapor R-200 (Büchi, Switzerland) at 70 C.
The remaining solid was extracted by methanol (2 · 1 mL),
the extract transferred to a test tube and the solvent
spontaneously evaporated at 21 C. Alternatively, an excess
of GPAO was added to 10 mL of 20 mM DAPY solution
in 20 mM potassium phosphate buffer, pH 7.0, and the
resulting mixture incubated at 37 C for 24 h. During that
time, GPAO was added twice more at 4-h intervals. After
ultrafiltration, the sample was processed as above.
RP-HPLC separation of DAPY oxidation product
The isolated DAPY oxidation product was dissolved in
0.3% (v/v) triethylamine acetate, pH 7.0. It was then
separated by RP-HPLC on a Supelcosil LC18 column,
25 cm · 4.6 mm i.d., 5 lm particles (Supelco, Bellefonte,
PA, USA) connected to a Gold Nouveau 125 NM HPLC
system equipped with a diode array detector Model 168
operating at 200–600 nm (Beckman, Fullerton, CA, USA).
The buffers used were as follows: A, 0.3% (v/v) triethylamine acetate, pH 7.0; B, 0.3% (v/v) triethylamine acetate,
pH 7.0, containing 60% (v/v) acetonitrile. Separations at
a flow rate of 1 mLÆmin)1 were run isocratically in the
FEBS 2004
4 Z. Lamplot et al. (Eur. J. Biochem.)
beginning (10 min), then with an increasing linear gradient
from 0 to 100% B for 20 min and isocratically at 100% B
for an additional 20 min. This was followed with a
decreasing linear gradient from 100 to 0% B in 8 min and
a short final isocratic step to give the total time 60 min.
Fractions showing highest absorption at 310 nm were
pooled, frozen and lyophilized. For MALDI-MS and
ESI-MS analyses, the obtained solids were extracted by
methanol; for 1H-NMR experiments the extraction was
performed using D2O.
MS of HPLC-separated DAPY oxidation product
MALDI-TOF-MS and MALDI-PSD-TOF-MS (PSD,
post source decay) were carried out using an Axima CFR
mass spectrometer (Kratos Analytical, Manchester, UK)
equipped with a nitrogen laser wavelength of 337 nm. Peak
power was 6.0 mW: positive mode with pulsed extraction
was used. MALDI probes were prepared by mixing 0.5 lL
of a sample diluted by acetonitrile with 0.5 lL of saturated
HABA in the same solvent. Acquired spectra were
processed by Kratos Axima CFR software KOMPACT
v. 2.1.1. Exact mass measurements to determine the
elemental composition of the DAPY oxidation product
were performed using ESI-Q-TOF-MS on a Q-Tof
microTM mass spectrometer (Micromass, Manchester,
UK). The collision-induced dissociation was used to get
MS/MS data. All samples were directly introduced to the
electrospray interface of the instrument by a syringe at a flow
rate of 5 lLÆmin)1. The ionization mode used produced
positively charged quasimolecular ions [M+H]+. Parameters of the electrospray were as follows: source voltage
2.5 kV, cone voltage 15 V, source temperature 80 C,
desolvation temperature 120 C. Acquired spectra were
processed by MASSLYNX v. 4 software (Micromass).
NMR spectroscopy of DAPY oxidation product
First, GPAO-catalyzed oxidation of DAPY was carried
out in 20 mM D2O-potassium phosphate buffer, pD 7.0,
similarly to previous work performed with a CAO and
agmatine [26]. DAPY.2HCl (3 mg) was dissolved in 0.5 mL
of D2O. Control 1H- and 13C-NMR spectra were recorded
at 27 C on a Bruker AVANCE 300 MHz NMR spectrometer (Bruker Analytik, Rheinstetten, Germany), using
tetramethylsilane as internal standard. After this measurement, the DAPY solution was pipetted into a test tube
containing a GPAO sample lyophilized from 0.5 mL of a
homogeneous GPAO (16 mgÆmL)1) in 20 mM potassium
phosphate buffer, pH 7.0. The mixture was shaken well on a
vortex and incubated at 30 C for 12 h. The enzyme protein
was then removed using the Microcon centrifugal cartridge
as described above, and the filtrate was used for recording
1
H- and 13C-NMR spectra. 1H-NMR spectra in D2O were
also measured with the DAPY oxidation product extracted
from a lyophilizate obtained after RP-HPLC separation of
the GPAO/DAPY reaction mixture.
Another NMR experiment was carried out as follows:
DAPY (5 mM) in 2 mL of 20 mM potassium phosphate
buffer, pH 7.0, was mixed with an excess of GPAO (5 mg,
added as a concentrated solution in the same buffer) and the
mixture was incubated at 30 C for 12 h. After that, the
same amount of GPAO was added again and the incubation
proceeded for an additional 12 h. Before 1H- and 13C-NMR
measurements, the resulting solution was ultrafiltered as
described above and the filtrate was lyophilized. The NMR
sample was prepared by extracting the lyophilizate with
0.5 mL of CD3OD.
Determination of free primary amino groups
Primary amino groups in GPAO were determined by
modification of the established TNBS method [27,28]. A
sample of the enzyme (0.1 mL of a buffered solution
containing 10–20 mgÆmL)1) was added to 0.9 mL of 4%
(w/v) sodium bicarbonate, pH 8.5, in a test tube and mixed
using a vortex. Later, 0.5 mL of 0.01% TNBS was added
with mixing and incubation at 40 C in the dark for 1 h.
ACA (1 mgÆmL)1) was used as a standard to construct the
corresponding calibration curve (10–50 lg). After incubation, all samples were measured at 345 nm against a blank
containing water instead of the enzyme. To determine free
amino groups in DAPY-reacted GPAO (100 : 1; 2 h of
incubation at 30 C), the reacted enzyme was exhaustively
dialyzed against 20 mM potassium phosphate, pH 7.0,
before an aliquot was processed as given above.
Chromatofocusing, quinone staining
Chromatofocusing was performed on a Mono P HR 5/20
column (Amersham Biosciences) connected to a BioLogic
Duo Flow liquid chromatograph (Bio-Rad, Hercules, CA,
USA). Loading buffer: 25 mM Tris/HCl, pH 8.2; elution
buffer: Polybuffer 96 (Amersham Biosciences, 3 mL) was
mixed with Polybuffer 74 (Amersham Biosciences, 7 mL),
diluted with water, adjusted to pH 5.0 with acetic acid and
then filled to a final volume of 100 mL. All samples were
dialyzed against the loading buffer before separation.
Redox-cycling quinone staining on nitrocellulose membrane
was carried out as described previously [29].
Results
Kinetic measurements
The oxidative conversion of DAPY was studied using two
plant CAOs after these enzymes had been isolated from
grass pea and sainfoin seedlings. Initial rates measured with
2.5 mM DAPY showed that the compound is a weak
substrate. For GPAO, the initial rate reached 5% of the
value measured for putrescine at the same concentration.
For OVAO, the initial rate with DAPY was 10% towards
that of cadaverine as the best substrate for this enzyme.
However, because OVAO prefers cadaverine to putrescine
by a factor 2.7 (and such a property is unique among plant
CAOs) [19], this value may be recalculated as 27% towards
that of putrescine.
Longer incubations of both studied enzymes with DAPY
led to a significant decrease in their catalytic activity toward
normal substrates. The inhibition was time- and concentration-dependent and irreversible, as the activity could not
be restored by dilution or dialysis. Pseudo-first-order
inhibition kinetics were observed at 30 C with DAPY
concentrations ranging from 5 to 40 lM; Fig. 1 shows
FEBS 2004
DAPY inactivates plant amine oxidases (Eur. J. Biochem.) 5
Fig. 1. Effect of incubation time on inactivation of GPAO by DAPY.
The semilogarithmic plot was constructed for the following DAPY
concentrations: 5 (j), 10 (m), 20 (r) and 40 lM (d). Activity was
measured with 70 nM enzyme in 0.1 M potassium phosphate buffer,
pH 7.0, at 30 C by means of the guaiacol spectrophotometric method
[21]. The inset shows the corresponding Kitz–Wilson replot for the
determination of kinact and KI values.
semilogarithmic plots for GPAO, where the slope for each
regression line represents the observed rate constant kobs.
The kinetic constants describing the inactivation of GPAO
and OVAO were determined from the corresponding Kitz–
Wilson replots (1/kobs vs. 1/[DAPY]; see inset in Fig. 1 as an
example). From these plots kinact, the maximal rate of
inactivation, is 1/y ) intercept, and KI, the concentration
required for half-maximal inactivation, is )1/x ) intercept.
The determined values for GPAO were similar when
measured in 0.1 M potassium phosphate or Bistris/HCl
buffers, pH 7.0 (Table 1). Comparatively, for OVAO,
inactivation by DAPY is slower, but the KI is lower.
GPAO and OVAO (both 70 nM in 0.1 M potassium
phosphate buffer, pH 7.0) were each individually incubated
with seven different concentrations of DAPY varying from
1 to 50 lM at 30 C for 1 h. Remaining activity was
determined by the ratio of the measured activity of the
inactivated enzyme to the control enzyme incubated without
DAPY. A plot of the remaining activity (%) vs. [DAPY]/
[GPAO] or [DAPY]/[OVAO] was constructed. Extrapolation of the linear portion of the data at lower [DAPY] gave
the partition ratio (turnover number minus one). This ratio,
the number of molecules leading to product per each
Table 1. Inactivation kinetics. Experiments were performed at 30 C in
0.1 M potassium phosphate buffer, pH 7.0, except where noted.
Inhibitor/enzyme
DAPY with GPAOb
DAPY with GPAO
DAPY with OVAO
N1-Methyl-DAPY
with GPAO
N5-Methyl-DAPY
with GPAO
a
t1/2 at saturationa
(min)
kinact
(min)1)
KI
(lM)
0.27
0.31
0.13
0.11
45
50
10
45
2.2
1.9
4.5
6.3
0.05
36
13.9
Time required for half of the enzyme to become inactivated in the
presence of saturating concentration of inhibitor. b In 0.1 M Bistris/
HCl buffer, pH 7.0.
Fig. 2. Partition ratio plot for inactivation of GPAO by DAPY.
Residual GPAO activities after 1 h of incubation with DAPY were
plotted against the corresponding values of the concentration ratio
[DAPY]/[GPAO]. Activity was measured with 70 nM enzyme in 0.1 M
potassium phosphate buffer, pH 7.0, at 30 C using the guaiacol
spectrophotometric method [21].
inactivation event, was determined to be 120 for DAPY/
GPAO (Fig. 2) and 200 for DAPY/OVAO.
The inhibition strength of DAPY is dependent on pH.
GPAO (70 nM) was incubated with 50 lM DAPY in 0.1
potassium phosphate buffers of different pH values over the
range 5.0–8.0 at 30 C for 1 h. The obtained remaining
activity values were then plotted against pH. DAPY showed
a maximal inhibition effect at pH 7.5. The extent of GPAO
inhibition by DAPY is also influenced by ionic strength.
The reaction was performed in 0.1 M Britton–Robinson
buffer, pH 7.2, where ionic strength had been adjusted with
KCl to reach values from the range 0.085–0.4. The
percentage of remaining activity after 1 h of incubation of
the reaction mixture (70 nM GPAO, 50 lM DAPY) at
30 C increases with increasing ionic strength (not shown).
Enzymes are protected against mechanism-based inhibitors by their substrates and competitive inhibitors. These
compounds bind at the active site and compete with binding
of the inhibitor. Inactivation of the enzyme is therefore
slowed down. GPAO (2 lM) was incubated with 100 lM
DAPY in the absence and presence of 1 mM cadaverine as a
substrate. At chosen time intervals, aliquots of the reaction
mixtures were taken out for activity assay. The protective
effect of cadaverine was significant. For example, after
15 min of incubation, the remaining activity was 15% in the
reaction mixture with cadaverine and only 8% without.
Due to the potential information that might be provided
about the mechanism of enzyme inactivation by DAPY, we
also determined the kinetics of inactivation of GPAO by the
two possible N-monomethyl analogs of DAPY. As shown
in Table 1, N1-methyl-DAPY and especially N5-methylDAPY were weaker inactivators relative to DAPY itself.
Spectrophotometry and spectrofluorimetry, TLC
Substrates of CAOs are known to disturb the characteristic
absorption spectrum of the enzymes [4]. Under anaerobiosis, the topaquinone cofactor maximum at 500 nm is
bleached after the substrate addition and replaced by a
complex spectrum of the Cu(I)-semiquinolamine radical
showing maxima at 360, 435 and 465 nm. This is supplemented with a peak at 315 nm that is thought to reflect the
6 Z. Lamplot et al. (Eur. J. Biochem.)
FEBS 2004
Fig. 3. Spectrophotometric studies on the
reactions of GPAO and OVAO with DAPY.
(Upper) Difference absorption spectrum of
GPAO (20 lM) after the addition of DAPY
(final concentration 1 mM) in air-saturated
0.1 M potassium phosphate buffer, pH 7.0.
The spectrum was recorded 2 s after mixing
the reactants at 30 C, using the rapid-scanning technique [9]. (Lower) Time-dependent
development of the DAPY oxidation product
as observed in difference absorption spectra.
The spectra were recorded using a solution of
OVAO (20 lM) in air-saturated 0.1 M potassium phosphate buffer, pH 7.0, after adding
0.1 M DAPY (1 mM final concentration) at
30 C. Intervals between scans: 15 s, total
time: 10 min.
presence of the aminoresorcinol form of topaquinone (the
fully reduced cofactor), which is in equilibrium. Using
rapid-scanning techniques, the mentioned spectral features
are observable also in the presence of air [9].
As shown in Fig. 3 (upper), rapid scanning after the
aerobic addition of DAPY to a purified GPAO in 0.1 M
potassium phosphate buffer, pH 7.0, revealed the formation
of a spectrum identical to that of a substrate-reduced CAO.
In addition, the reaction of the studied plant CAOs with
DAPY gave rise to an oxidation product providing near
UV/visual absorption with a maximum at 310–315 nm,
which increased in intensity with temperature. Absorbances
measured after 90 min of incubation of GPAO/DAPY
reaction mixtures at 50 C were almost three times higher
than those measured after the incubation at 37 C. Figure 3
(lower), shows an increasing development of the product
within the first 10 min after mixing OVAO with DAPY in
0.1 M potassium phosphate buffer, pH 7.0. The same
spectrum was also observable in the GPAO/DAPY reaction
mixture. In 0.1 M Bistris/HCl buffer, pH 7.0, the product
absorption maximum was shifted to 325 nm (not shown).
GPAO and OVAO were fully inactivated by incubation
with an excess of DAPY and no activity could be recovered
by dialysis. The inactivated enzymes after dialysis were still
faint yellow due to a broad absorption below 330 nm, but
the color intensity was substantially decreased.
After separation of the enzyme protein by ultrafiltration,
the GPAO/DAPY reaction mixture exhibited a fluorescence
emission spectrum with a maximum at 445 nm (shoulders at
485 and 520 nm) when excited at 310 nm. For model
compounds, the following fluorescence characteristics were
obtained: DDD, solvent water, emission maximum at
503 nm (excitation at 410 nm); 3-hydroxypyridine, solvent
water, emission maximum at 460 nm (excitation at
310 nm); NADH, solvent water, emission maximum at
465 nm (excitation at 340 nm); pyrrole, solvent water,
emission maximum at 360 nm (excitation at 290 nm).
TLC experiments revealed the presence of a free primary
amino group in the DAPY oxidation product obtained by
the reaction of GPAO (positive ninhydrin spot, Rf ¼ 0.62);
DAPY itself showed Rf ¼ 0.33 in the same system. Staining
for tertiary amines (Draggendorff’s reagent) was also
positive for the GPAO/DAPY reaction mixture (an orange
spot, Rf ¼ 0.62). Staining for aldehydes using Schiff’s
reagent was negative.
Colorimetric detections of DAPY oxidation product
Kovacs’ reagent containing DMAB (detects indoles and
pyrroles [25]) was previously used for the visualization of
pyrrole derivatives formed by the oxidation of DABY by
plant amine oxidases [7,8]. Reaction mixtures of the studied
CAOs with DAPY reacted positively with Kovacs’ reagent
after a period of incubation and provided a red soluble
adduct upon heating. The red color intensity increased with
increasing temperature in the range 30–50 C. A typical
absorption spectrum of the adduct with a maximum at
520 nm (shoulder at 500 nm) is shown in Fig. 4 (upper).
FEBS 2004
DAPY inactivates plant amine oxidases (Eur. J. Biochem.) 7
to form the corresponding aminoaldehydes, which spontaneously cyclize to 1-pyrroline and 1-piperideine, respectively. The latter cyclic imines condense with ABA to
generate the corresponding substituted dihydroquinazolinium compounds. The DAPY oxidation product obtained
by the reaction of GPAO provided an adduct with ABA
characterized by an absorption maximum at 430 nm (not
shown).
The ninhydrin reagent described for activity assay of
CAOs by Naik et al. [24] was also tested to trap the DAPY
oxidation product in the reaction mixture with GPAO. The
same cyclic imines above (1-pyrroline and 1-piperideine)
react with ninhydrin in strongly acidic medium to form
colored compounds of unknown structure with absorption
maxima at 440 and 515 nm, respectively [30]. The DAPY
oxidation product displayed a broad absorption between
400 and 550 nm with a maximum at 465 nm after reaction
with ninhydrin (not shown).
MS of DAPY reaction mixture
Fig. 4. Reaction of the DAPY oxidation product and a dihydropyridine
model compound with 4-(dimethylamino)benzaldehyde. (Upper) An
aliquot (1 mL) of the GPAO/DAPY reaction mixture in 0.1 M
potassium phosphate buffer, pH 7.0, was mixed with 2 mL of Kovacs’
reagent, incubated at 50 C for 30 min and finally cooled in an icebath. The absorption spectrum was recorded against a blank containing water instead of the reaction mixture; (upper) reaction mixture
(lower) reaction mixture after removing protein by ultrafiltration. For
experimental details see Materials and methods. (Lower) A 1 mL
portion of 5 mM DDD was mixed with 2 mL of Kovacs’ reagent,
incubated at 50 C for 30 min and finally cooled in an ice bath.
Absorption spectra were then recorded against a blank containing
water instead of the dihydropyridine.
Replacing DMAB in Kovacs’ reagent with DMAC led to a
shift of the adduct absorption maximum to 650 nm (not
shown). However, if the reaction mixture was dialyzed
before the addition of the reagent, the spectrum was almost
negligible (Fig. 4, upper). Three model compounds were
tested for this reaction. The synthesized DDD reacted with
Kovacs’ reagent to form a product with an absorption
maximum at 600 nm having a shoulder at 560 nm (Fig. 4,
lower). NADH also reacted with the reagent and provided a
spectrum with a single peak centered at 510 nm. DMABreacted pyrrole provided a maximum at 565 nm with a
shoulder at 520 nm.
The use of ABA for the spectrophotometric activity assay
of plant CAOs was refined almost four decades ago [23].
Plant CAOs oxidize the diamines putrescine and cadaverine
Figure 5 (upper) shows an ESI-IT mass spectrum of the
GPAO/DAPY reaction mixture prepared using 0.1 M
ammonium bicarbonate, pH 7.8. There are two major
peaks of the reaction product observable in the spectrum
with m/z 178.3 and 222.3. The former ion showed fragment
peaks with m/z 161.3, 149.2 and 135.2, the latter provided
peaks with m/z 205.2, 193.2 and 176.2 in the respective
MS/MS spectra. The peak with m/z 222.3 was not observed
when the reaction was carried out in 0.1 M Bistris/HCl
buffer, pH 7.0. Figure 5 (lower) shows a mass spectrum of
the GPAO/DAPY reaction mixture prepared in 0.1 M
ammonium bicarbonate containing ACA as a reagent for
trapping of the product aminoaldehyde, where several new
peaks appeared. ACA itself is represented by a peak with
m/z 132.1 (MS/MS: a clear fragment peak with m/z 114.1.)
There is one more peak visible with m/z 211.3 (MS/MS:
fragment peaks with m/z 193.3, 106.0 and 96.0), which
probably reflects an adduct of the reaction product with
ACA.
ESI-IT-MS of the low molecular mass fraction of the
GPAO/DAPY reaction mixture prepared in 20 mM
potassium phosphate buffer, pD 7.0 (made in D2O for
the purpose of NMR spectroscopic analysis) revealed
isotopic peaks belonging to quasimolecular ions of the
reaction product. The highest intensity was observed for
a peak with m/z 179.2, lower intensities were observed
for peaks in the following order: m/z 181.2, 180.2, 182.2
and 178.2. The peak with m/z 179.2 provided an MS/MS
spectrum showing fragments with m/z 162.2, 150.2 and
136.2 (not shown).
HPLC separation and MS analysis of DAPY oxidation
product
HPLC separation of the isolated DAPY oxidation product
from enzymatic microscale production was carried out
using an instrument equipped with a diode array detector.
Thus individual runs could be monitored continuously at
214, 240 and 310 nm. The buffer system used was chosen
according to that published for peptide separation from
tryptic digests [31].
8 Z. Lamplot et al. (Eur. J. Biochem.)
FEBS 2004
Fig. 5. ESI-IT-MS analyses of GPAO/DAPY
reaction mixtures. (Upper) DAPY (5 mM) in
0.1 M ammonium bicarbonate, pH 7.8, was
mixed with an excess of GPAO and incubated
at 30 C for 24 h. After removing protein by
ultrafiltration, the reaction mixture was analyzed by ESI-IT-MS as described in Materials
and methods. (Lower) A combined solution of
DAPY and ACA (each 5 mM) in 0.1 M
ammonium bicarbonate, pH 7.8, was mixed
with an excess of GPAO and incubated at
30 C for 24 h. After removing protein by
ultrafiltration, the reaction mixture was analyzed by ESI-IT-MS as described in Materials
and methods.
Three peaks at elution times 3.0 min (1), 15.8 min (2) and
23.0 min (3) were collected and their composition analyzed
using ESI-MS and MALDI-MS. The largest peak absorbing at 310 nm (peak 1) appeared to correspond to a single
chemical compound (m/z 178.1). The corresponding
ESI-IT-MS/MS spectrum is presented in Fig. 6, where
Fig. 6. MS/MS spectrum of DAPY oxidation product. The isolated
DAPY oxidation product from enzymatic microscale production was
dissolved in 0.3% (v/v) triethylamine acetate, pH 7.0, and separated by
RP-HPLC as described in Materials and methods. The 310 nm-peak
at an elution time 3.0 min was collected and analyzed by ESI-IT-MS
and MS/MS. The spectrum shown was recorded after collisioninduced fragmentation of the parent ion belonging to the DAPY
oxidation product (m/z 178.1).
fragmentation peaks were observed with m/z 161.1, 149.1,
144.1, 135.1, 132.1, 120.1, 109.1, 95.0 and 82.0. After
lyophilization, a solid obtained from peak 1 did not produce
irreversible inhibition of the studied enzymes. There was one
more compound in peak 2 with m/z 257.1, whose MS/MS
spectrum provided peaks with m/z 240.1, 228.1, 214.3,
202.3, 176.2, 161.2, 149.2 and 133.0. Finally, peak 3
contained at least five compounds. In addition to those
with m/z 178.1 and 257.1 there were three more peaks with
m/z 334.3 (fragmentation: m/z 317.3, 305.2, 291.1, 253.2 and
240.1), 350.2 (fragmentation: m/z 333.2 and 307.1) and
431.3 (fragmentation: m/z 414.3, 388.3 and 337.1).
MALDI-TOF-MS of the separated peak 1 provided a
single compound with m/z 178.1; the same m/z value was
obtained by an ionization without using the HABA matrix.
MALDI-PSD-TOF-MS provided a fragmentation pattern
consistent with the ESI-IT-MS/MS experiments already
mentioned (data not shown).
ESI-Q-TOF-MS analysis of the HPLC peak 1 permitted
the determination of both exact mass and elemental
composition of the DAPY oxidation product. An m/z
value of 178.14 was obtained, which matches a molecular
formula C10H16N3. Peaks in the corresponding MS/MS
spectrum provided the following m/z values and elemental
composition of ions: 161.11 (C10H13N2), 149.11 (C9H13N2),
135.09 (C8H11N2), 109.08 (C6H9N2), 95.06 (C5H7N2) and
82.06 (C5H8N).
FEBS 2004
DAPY inactivates plant amine oxidases (Eur. J. Biochem.) 9
Fig. 7. 1H-NMR spectrum of DAPY oxidation product. DAPY (5 mM) in 2 mL of 20 mM potassium phosphate buffer, pH 7.0, was mixed with an
excess of GPAO (5 mg, added as a concentrated solution in the same buffer) and the mixture was incubated at 30 C for 12 h. The same amount of
GPAO was added again and the incubation proceeded for an additional 12 h. The resulting solution was centrifuged to remove protein precipitate
and ultrafiltered, and the filtrate was lyophilized. The NMR sample was finally prepared by extracting the lyophilizate with 0.5 mL of CD3OD. The
insets shows a detailed view of the vinylic doublet signals belonging to 2,3-dihydropyridine.
For initial experiments, DAPY oxidation by GPAO was
performed in 20 mM potassium phosphate buffer made in
D2O (pD 7.0). The enzyme protein was removed by
ultrafiltration and the filtrate was directly measured. Several
signals with the following chemical shifts were observed in
the 13C-NMR spectrum: d (p.p.m.) 17.0, 25.1, 29.1, 37.9,
62.5, 72.1, 74.3, 82.7, 123.0, 131.2 and 159.8. The corresponding 1H-NMR spectrum contained various signals in
the region 2.0–4.5 p.p.m., but these were largely obscured
by the residual water peak (4.5–5.0 p.p.m.). In addition, two
vinylic signals at d 5.7 and 7.8 were observed, but their
intensities were too small to ascertain their multiplicities
(not shown). Much better 1H-NMR spectra were obtained
after the extraction of the ultrafiltered and lyophilized
GPAO/DAPY reaction mixture by CD3OD. Figure 7
shows such a spectrum, including the following signals: d
2.63–2.73 (m), 3.12 (q, J ¼ 6.95 Hz), 3.31 (p, J ¼ 1.65 Hz),
3.79 (t, J ¼ 2.20 Hz), 4.19 (t, J ¼ 2.20 Hz), 5.35 (d, J ¼
6.22 Hz), 7.82 (d, J ¼ 6.22 Hz). The spectrum is partially
obscured by two signals of residual methanol at d 3.3 and
4.6–5.1. There are also three complex signals that are quite
difficult to interpret, which are centered at d values of 2.80,
3.55 and 3.65. 13C-NMR spectra measured with the GPAO/
DAPY mixture in CD3OD resembled those recorded in
D2O, but the obtained quality was lower. 1H-NMR spectra
were also recorded in D2O using the solid obtained by
lyophilization of the peak 1 from the HPLC separation
mentioned above. However, NMR signal intensities were
insufficient due to the low concentration of compound. In
addition, these spectra were obscured by two peaks of
residual triethylamine from the elution buffer at d 1.2 and
3.2 (data not shown).
formed by the turnover of acetylenic diamine substrates
[7–11]. The cDNA of GPAO subunit (without the signal
peptide) has been cloned and sequenced (D. Kopečný,
N. Houba-Hérin, H. G. Faulhammer & M. Šebela,
unpublished results; EMBL/GenBank accession number
AJ786401). The translated protein sequence is largely
similar to those two published for PSAO [32,33] and
comprises 38 lysines. GPAO and PSAO have very similar
peptide maps obtained by MALDI-MS experiments [34].
PSAO protein sequence of Koyanagi et al. [32], which is
deposited under accession number JC7251 (NCBI Protein
Databank) differs slightly from that of Tipping and
McPherson [33], accession number Q43077, in that whereas
the former sequence comprises 39 lysine residues, the latter
has only 38 lysine residues, as calculated for the mature
form of the protein. Although native PSAO (a dimer) is thus
expected to comprise 76–78 lysine residues, only 32 are
solvent-accessible [8]. We determined 38 accessible lysines
per dimer in the native GPAO using a modified protocol
with the TNBS reagent, and 36 lysines per dimer (average
values from repeated measurements) after reaction with the
DAPY.
Quinone-staining experiments [29] with the DAPY-inactivated GPAO were positive and demonstrated that the
topaquinone cofactor was not modified in a redox-inactive
form (data not shown). Chromatofocusing experiments
performed according to that with DABY-inactivated GPAO
[8] revealed that the pI value of the DAPY-inactivated
GPAO was not dramatically changed. The native GPAO
is characterized by a pI of 7.2 [18]. After the reaction with
an excess of DAPY, the enzyme sample comprised more
species having isoelectric points of pI 6.8–7.5 (Fig. 8).
Therefore, the inactivation resulted in a heterogeneous
mixture of differently charged protein molecules.
Other analyses
Discussion
Lysine residues in plant CAOs are possible targets for
covalent binding of reactive electrophilic aminoaldehydes
DAPY was synthesized as an analog of cadaverine
(pentane-1,5-diamine), which is known as the best substrate
NMR spectroscopy of DAPY oxidation product
10 Z. Lamplot et al. (Eur. J. Biochem.)
Fig. 8. Chromatofocusing of DAPY-inactivated GPAO. Chromatofocusing was performed on a Mono P HR 5/20 column using a BioLogic
Duo Flow liquid chromatograph at a flow rate of 1 mLÆmin)1. The
loading buffer was 25 mM Tris/HCl, pH 8.2, and the elution buffer was
a diluted mixture of Polybuffer 96 and Polybuffer 74 adjusted to
pH 5.0 with acetic acid. All samples were dialyzed against the loading
buffer before separation. Approximately 5 mg of protein was loaded.
of plant CAOs [4]. Contrary to naturally occurring diamines, the DAPY molecule contains a triple bond at the
b- and c-positions from the two primary amine termini. The
oxidative conversion of the compound by GPAO and
OVAO was demonstrated by measuring the production of
H2O2 using spectrophotometry. Therefore, the enzymes are
able to undergo complete turnover [3]. However, DAPY
was oxidized more efficiently by OVAO than by GPAO.
Although, to date, OVAO has not been crystallized nor had
its structure solved, this observation might be explained in
terms of different arrangements of the active sites of the
enzymes resulting in the preference for C5-diamine substrates by OVAO.
Similarly to the conversion of its lower homolog DABY
by PSAO [7], DAPY oxidation by the studied enzymes led
to their irreversible inhibition. The apparent inactivation
constants KI of 10)5 M are on the same order of magnitude
as those KI values previously described for BEA [6] and
DABY [7] in the reactions with lentil seedling amine oxidase
(LSAO) and PSAO, respectively. The obtained rates of
inactivation resembled for example that for BEA as
measured with LSAO [6], but they were lower than that
for DABY in the reaction with PSAO [7]. At the same time,
whereas the determined partition ratio values with GPAO
and OVAO were in the range observed for BEA (r ¼ 100)
and some other monohalogenated alkylamines (r < 500) in
the reactions with LSAO [6], they were significantly higher
than that for DABY and PSAO (r ¼ 17) [7]. From this
point of view, plant CAOs are more resistant to the
inactivation by DAPY than by DABY. Binding of DAPY
at the active site of GPAO is dependent on both pH and
ionic strength. In these features, the reaction does not differ
from those of typical plant CAO substrates like putrescine
or cadaverine. GPAO and OVAO inactivation caused by
the substrate DAPY fulfills the criteria of a mechanismbased inhibition: it is time dependent, irreversible and can be
weakened in the presence of a normal substrate [5,7,11,22].
DAPY oxidations by GPAO and OVAO were accompanied by spectral changes. The typical absorption spectrum of a substrate-reduced CAO, which appeared after the
rapid addition of DAPY to either GPAO or OVAO
solutions, was in accordance with the substrate properties of
DAPY determined by the guaiacol spectrophotometric
FEBS 2004
assay. Presuming that DAPY oxidation follows the same
mechanism as for common substrates of plant CAOs, the
reaction should generate 5-amino-2-pentynal or 5-amino-3pentynal as a product aldehyde (DAPY is not a symmetric
molecule). Although no free aldehyde was detected by TLC
in the reaction mixture, indirect evidence for an aminopentynal turnover product was that an adduct formed
(m/z 211.3) when DAPY was enzymatically oxidized in the
presence of ACA. This adduct exhibited a MS/MS
fragmentation pattern similar to that of free ACA, showing
a loss of a water molecule from the carboxylic group ()18,
m/z 211.3 fi m/z 193.3).
There are detailed reports on the mechanism-based
inhibition of CAOs by DABY in the literature [7–11]. The
authors have shown that DABY is oxidized to 4-amino-2butynal, which induces inactivation by adducting to a
nucleophile in the substrate channel. In addition, the
reaction brings about multiple surface labeling of the
enzyme, which probably occurs through solvent-accessible
nucleophilic residues [8]. DAPY oxidation appears to result
in much less extensive protein modification, as the number
of free primary amino groups in the enzyme did not change
dramatically. Chromatofocusing of DAPY-inactivated
GPAO revealed only a small change in the isoelectric point,
likely caused by the modification of a few amino acid
residues upon binding of aminopentynal. This binding
seems to be nonspecific, as the existence of some microheterogeneity (at least two species with different pI values)
in the inactivated enzyme was confirmed. As in the case of
DABY, the cofactor topaquinone is not modified by the
reaction, as demonstrated by an unchanged quinone redox
staining.
Absorption spectroscopy demonstrated the formation of
a secondary product in the GPAO/DAPY reaction mixture
with kmax at 310 nm and emitted fluorescence (kmax at
445 nm) upon excitation at 310 nm, supportive of extended
conjugation. Dialysis of the reaction mixtures containing
GPAO or OVAO and DAPY, resulted in decoloration,
demonstrating that the chromophore generated is a free low
molecular mass compound. Several colorimetric assays
provided evidence for the presence of a nitrogenous
heterocycle, in addition to a free amino group. The
GPAO/DAPY reaction mixture exhibited a positive reaction with ABA and ninhydrin reagents, similar to that
observed for the cyclic imines 1-pyrroline and 1-piperideine
formed upon enzymative oxidation of putrescine and
cadaverine, respectively. In acidic medium, DMAB
and DMAC reacted with the DAPY oxidation product
(and also with the model compounds DDD and NADH) to
give markedly colored adducts. This probably occurs upon
binding of the reagents at the a-position of the heterocycle
[8].
The rigidity of the triple bond in the presumed aminopentynal turnover product would prevent cyclization, but
this geometrical constraint would be relaxed by conjugate
addition of a nucleophile. Thus, as shown in Fig. 9, if a
molecule of unreacted DAPY is added to either of the two
possible aminopentynal turnover products, cyclization to a
six-membered heterocycle and eventual formation of a
common resonance-stabilized 4-amino-2,3-dihydropyridine
would be predicted. The extended conjugation would be
consistent with the observed absorption and fluorescence
FEBS 2004
Fig. 9. Mechanism of DAPY oxidation by GPAO. The scheme reflects
the summary results of kinetic, spectrophotometric, spectrofluorimetric, MS and NMR experiments performed in this study. First, DAPY
is oxidized by the enzyme to either 5-amino-2-pentynal or 5-amino-3pentynal, which subsequently (after tautomerization in the latter case)
reacts with a second DAPY molecule (or ACA) acting as nucleophile.
After nucleophilic adduction, the two possible aldehydes undergo
cyclization to form ultimately the same resonance stabilized 4-amino2,3-dihydropyridine ring. Enzyme inactivation may follow the same
overall reaction except that the 4-amino-2,3-dihydropyridine is formed
on the enzyme by adduction of the intial aminopentynal turnover
product to a lysine residue.
spectral properties. The final structure is also consistent with
MS and NMR data. In particular, the two coupled vinyl
doublets at 5.35 and 7.82 p.p.m. in the 1H-NMR spectrum
(Fig. 7) are consistent with the electron-rich C5 and
electron-deficient C6 positions of the 4-amino-2,3-dihydropyridine. The 13C-NMR spectrum additionally demonstrated the presence of a triple bond (d 74.3 and 82.7 p.p.m) and
aliphatic carbon atoms (signals of d 20–40 p.p.m), consistent with presence of an unoxidized DAPY moiety.
The theoretical molecular mass calculated for the predicted N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was in
accordance with that determined by ESI-Q-TOF-MS
(177.14 Da) and the fragmentation pattern of the peak
with m/z 178.1 observed by MALDI-MS/ESI-MS. Either
the N1 or N5 amino group of unoxidized DAPY could add
to the initial aminopentynal turnover product. Although an
insufficient amount of the oxidation product was available
to perform the detailed two-dimensional NMR experiments
that would be needed to distinguish between the N1- and N5(2,3-dihydropyridin-4-yl)-1,5-diamino-2-pentyne isomers,
the observed ESI-IT-MS/MS fragmentation peaks (Fig. 6)
are most consistent with the former compound, as depicted
in Fig. 10. A small peak at m/z 144.1 (C9H8N2) requires
substantial dehydrogenation and is hard to reconcile with a
particular structure. It should be pointed out that only the
peaks with m/z 135.1 and 132.1 are consistent with only the
N1- and not the N5-isomer, and a small peak at m/z 120.1
(C7H8N2) is most readily reconciled with the N5-isomer.
Thus, we cannot exclude the possibility that the DAPY
oxidation product represents a mixture of mainly N1-(2,3dihydropyridin-4-yl)-1,5-diamino-2-pentyne, contaminated
with a small amount of the N5-isomer that coelutes during
RP-HPLC separation.
The MS experiments performed in the present study also
demonstrated that the aminopentynal product undergoes
further oligomerization reactions, which result in the
formation of compounds having higher molecular masses
(256–430 Da). For example, the peak with m/z 257.1
DAPY inactivates plant amine oxidases (Eur. J. Biochem.) 11
Fig. 10. MS/MS fragmentation scheme for DAPY oxidation product.
The scheme reflects the postulated collision-induced dissociation of
the parent DAPY oxidation product ion (m/z 178.1) assigned as
N1-(2,3-dihydropyridin-4-yl)-1,5-diamino-2-pentyne, according to the
observed MS/MS peaks obtained by ESI-IT-MS/MS (Fig. 6).
probably represents adduction of aminopentynal to both
amino groups of the same unoxidized DAPY molecule
to give N1,N5-bis-(2,3-dihydropyridin-4-yl)-1,5-diamino-2pentyne, and the peak with m/z 334.1 suggests even one
more aminopentynal coupling. Finally, it should be pointed
out that the 4-amino-2,3-dihydropyridine core structure is
consistent with the molecular ion m/z 211.3 observed for the
ACA-trapped DAPY oxidation product, wherein ACA
rather than unoxidized DAPY would add to the initial
aminopentynal turnover product (see Fig. 9).
According to the mechanism for formation of the
chromophoric DAPY oxidation product (Fig. 9), the same
resonance-stabilized 4-amino-2,3-dihydropyridine moiety
would form if the initial aminopentynal turnover product
were trapped by an enzyme-based lysyl residue (Fig. 9). The
evidence for modification of at least some lysines during
incubation of GPAO with DAPY suggests that such
adduction could be responsible for the irreversible enzyme
inactivation observed. In this regard, the inactivation
mechanism would then be highly analogous to that
discerned for inactivation of PSAO or GPAO by DABY,
where the initial 4-amino-2-butynal product undergoes
conjugate addition by a channel lysyl residue, followed by
dehydrative cyclization to give a 3-aminopyrrole [8]. On the
basis of the highly apparent formation of the low molecular
mass DAPY oxidation product identified here, one might
speculate why the analogous product, N-(pyrrole-3-yl)-1,4diamino-2-butyne, was not observed during plant CAO
metabolism of DABY. Although such product might have
actually been present, there are two reasons why it is
probably less apparent. The first is that DABY is a more
potent inhibitor than DAPY, so that higher concentrations
of the latter, amenable to formation of the observed
coupling product, were employed. The second is that the
aminopentynal turnover product from DAPY appears to
modify the pertinent substrate channel lysine with significantly less efficiency than does the 4-amino-2-butynal
product from DABY, so that there is greater turnover
prior to inactivation.
To obtain clues as to the mechanism of inactivation of
GPAO by DAPY, we investigated the inhibitory potency of
the two possible mono-N-methyl derivatives of DAPY with
FEBS 2004
12 Z. Lamplot et al. (Eur. J. Biochem.)
this enzyme. Topaquinone-dependent enzymes are mostly
not known to metabolize secondary amines. The finding
that one but not the other of the two N-methyl derivatives of
DAPY acted as an inactivator, would suggest that metabolism leading to inactivation occurs only at the propargylamine terminus or the homopropargylamine terminus.
Our finding that both derivatives act as inactivators of
GPAO, albeit weaker than DAPY, suggests that enzyme
metabolism of DAPY leading to inactivation can occur at
either amino group, as shown in Fig. 9.
In conclusion, DAPY was found here to be both a
substrate and inactivator of plant CAOs. Prior to complete
enzyme inactivation, the enzymes generate significant
amounts of two possible aminopentynal turnover products.
Either 5-amino-3-pentynal (after tautomerization) or
5-amino-2-pentynal can condense with a molecule of
unoxidized DAPY to give an adduct capable of cyclization
to the same 4-amino-2,3-dihydropyridine, which is apparently resistant to hydrolysis on account of extended
resonance stabilization. The structure of the final product,
most likely N1- rather than N5-(2,3-dihydropyridin-4-yl)1,5-diamino-2-pentyne, was supported by spectrophotometric, spectrofluorimetric, MS and NMR measurements.
Elucidation of the adduct structure suggests that enzyme
inactivation occurs through the same chemical mechanism,
but involving an enzyme-based lysyl residue, rather than a
second DAPY molecule, in adduct formation with the
initial aminopentynal turnover product (Fig. 9). Nonetheless, further studies are needed to ascertain the true
molecular nature of enzyme modification leading to inactivation by DAPY. Although DAPY is not such a powerful
inactivator in comparison with the previously studied
shorter analog DABY, the reaction with DAPY does not
result in as extensive labeling of the enzyme as was observed
for DABY. The significance of the present work resides also
in the possible use of plant CAOs for organic synthesis,
which has already been suggested [35]. Pure chemical
preparation of a dihydropyridine such as that characterized
here would be difficult.
Acknowledgements
This work was supported by grants MSM 153100010, MSM 153100013
and 14BI3 (Czech-Italian cooperation, a joint grant with the Ministry
of Foreign Affairs, Italy) from the Ministry of Education, Youth and
Sports, Czech Republic, and by grant GM 48812 from the National
Institutes of Health (to L.M.S.). Fluorescence spectra were recorded by
courtesy of Dr Martin Modrianský from the Institute of Medical
Chemistry and Biochemistry, Faculty of Medicine, Palacký University.
Dr Ivo Frébort, Palacký University, is thanked for the initial impetus to
start with this research.
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