1,5-Diamino-2-pentyne is both a substrate and inactivator of plant
copper amine oxidases
Zbyne
ˇ
k Lamplot
1
, Marek S
ˇ
ebela
1
, Michal Malon
ˇ
2
, Rene
´
Lenobel
2
, Karel Lemr
3
, Jan Havlis
ˇ
4
, Pavel Pec
ˇ
1
,
Chunhua Qiao
5
and Lawrence M. Sayre
5
1

1
-methyl and N
5
-methyl
analogsofDAPYweretestedwithGPAOandwereweaker
inactivators (especially the N
5
-methyl) than DAPY. Pro-
longed incubations of GPAO or OVAO with DAPY resul-
ted i n the appear ance of a yellow–brown c hromophore
(k
max
¼ 310–325 nm depending on the w orking buffer) .
Excitation at 310 nm was associated with emitted fluores-
cence with a maximum at 445 nm, suggestive of extended
conjugation. After dialysis, the color intensity was substan-
tially decreased, indicating the f ormation of a low molecular
mass secondary product of turnover. The compound pro-
vided positive reactions with ninhydrin, 2-aminobenzalde-
hyde and Kovacs’ reagents, suggesting the presence of an
amino group and a nitrogen-con taining heterocyclic struc-
ture. The secondary product was separated chromato-
graphically and was found not to irreversibly inhibit GPAO.
MS indicated an exact molecular mass (177.14 Da) and
molecular formula (C
10
H
15
N
3

oxidative deamination is conversion of the initial substrate
Schiff base (quinoimine) to a product Schiff base (quino-
aldimine) facilitated by Ca proton abs traction 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 H
2
O
2
and NH
4
+
. 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 r ole o f copper i n the
reoxidation step h as not been sufficiently elucidated for
Correspondence to M. S
ˇ
ebela, Department of Biochemistry, Faculty of
Science, Palacky´ University, S
ˇ
lechtitelu˚ 11, CZ-783 71 Olomouc,
Czech Republic. Fax: + 420 5856 34933; Tel.: + 420 5856 34927;
E-mail:
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-pen-
tyne; DDD, 3,5-diacetyl-2,6-dimethyl-1,4-dihydropyridine; DMAB,

(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 o f 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 concom-
itant inactivation. However, in comparison with the effect of
DABY previously published [8], the modification extent is
considerably decreased. Only a few amino acid side c hains
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

dine, pyrrole and 2,4,6-trinitrobenzenesulfonic acid
(TNBS) solution (5%, w/v) were from Sigma (St. Louis,
MO, USA). Deuterium oxide (D
2
O, 99.96%) and d
4
-
methanol (CD
3
OD, 99.95%) were from Aldrich (Milwau-
kee, WI, USA). 6-Aminocaproic acid (ACA) and NADH
were supplied by Fluka (Buchs, Switzerland). 2-(4-Hy-
droxyphenylazo)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)seed-
lings were prepared in homogeneous forms following
published protocols [18,19]. Specific activities assayed with
cadaverine as a substrate were 50 and 120 UÆmg
)1
, respect-
ively. Bovine liver catalase (2000 UÆmg
)1
) and horseradish
peroxidase (100 UÆmg
)1
) were commercial products from
Fluka. Protein content in enzyme samples was estimated

Ó FEBS 2004 DAPY inactivates plant amine oxidases (Eur. J. Biochem. 271) 4697
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 rea-
gents, respectively. Aldehyde groups were detected using
Schiff’s reagent.
Spectrofluorimetry
AsolutionofDAPY(5m
M
)in20m
M
potassium phos-
phate 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 spectroflu-
orimeter (Perkin–Elmer, Boston, MA, USA). The oxidized
DAPY was measured with a fixed excitation at 310 n m.
Similarly, for the model compounds, the respective wave-
lengths 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

M
DAPY; i nitial
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 Microc on c entrifugal 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
5m
M
buffered DAPY solution with an excess of GPAO.
Two buffers were used to optimize results: 0.1
M
ammo-
nium bicarbonate, pH 7.8, and 0.1
M
Bistris/HCl, pH 7.0.
To evaluate the reactivity of the initial product aminoalde-
hyde, the reaction was also carried out in the presence of
5m

washed with the same buffer. Then 50 mL of 5 m
M
DAPY
in 10 m
M
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 m
M
ammonium bicarbonate, and the eluate w as 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 (Bu
¨
chi, Switzerland) at 70 °C.
The remaining solid was extracted by methanol (2 · 1mL),
the extract transferred to a test tube and the solvent
spontaneously evaporated at 21 °C. Alternatively, an excess
of GP AO was added to 10 mL of 20 m
M
DAPY solution
in 20 m
M
potassium phosphate buffer, pH 7.0, and the

H-NMR experiments the extraction w as
performed using D
2
O.
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 n m. 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 K ratos A xima CFR software
KOMPACT
v. 2.1.1. Exact m ass measurements to determine the
elemental composition of the DAPY oxidation product
were performed using ESI-Q-TOF-MS on a Q-Tof
micro
TM
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]
+
. Parame-

M
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 t he filtrate was used for recording
1
H- and
13
C-NMR spectra.
1
H-NMR spectra in D
2
Owere
also measured with the DAPY oxidation product extracted
from a lyophilizate obtained after RP-HPLC separation of
the GPAO/DAPY reac tion mixture.
Another NMR experiment was carried out as follows:
DAPY (5 m
M
)in2mLof20m
M
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 a dditional 12 h. Before
1
H- and

Chromatofocusing, quinone staining
Chromatofocusing was p erformed 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 m
M
Tris/HCl, pH 8 .2; elution
buffer: Polybuffer 96 (Amersham Biosciences, 3 mL) was
mixed with Polybuffer 74 (Amersham Biosciences, 7 m L),
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 b efore 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 m
M
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.

buffers, pH 7.0 (Table 1). Comparat ively, for OVAO,
inactivation by DAPY is slower, but the K
I
is lower.
GPAO and OVAO (both 70 n
M
in 0.1
M
potassium
phosphate buffer, pH 7.0) were each individually incubated
with seven different concentrations of DAPY varying from
1to50l
M
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 (%) v s. [DAPY]/
[GPAO] or [DAPY]/[OVAO] was constructed. Extrapola-
tion 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
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 n
M
) was incubated with 50 l
M
DAPY in 0.1
potassium phosphate buffers of different pH values over the

15 min of incu bation, 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 b y the
two p ossible N-monomethyl analogs o f DAPY. As shown
in Table 1, N
1
-methyl-DAPY and especially N
5
-methyl-
DAPY 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 anaero-
biosis, 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 supple-
mented with a peak at 315 nm that is thought to reflect the
Fig.1. EffectofincubationtimeoninactivationofGPAObyDAPY.
The semilogarithmic plot was constructed for the following DAPY
concentration s: 5 (j), 10 (m), 20 (r)and40l
M
(d). Activity was
measured with 70 n
M
enzyme in 0.1
M
potassium phosphate bu ffer,

DAPY with GPAO 0.31 50 1.9
DAPY with OVAO 0.13 10 4.5
N
1
-Methyl-DAPY
with GPAO
0.11 45 6.3
N
5
-Methyl-DAPY
with GPAO
0.05 36 13.9
a
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 G PAO by DAPY.
Residual GPAO ac tivities after 1 h of incubation with D APY were
plotted against the corresponding values of the concentration ratio
[DAPY]/[GPAO]. Activity was measured with 70 n
M
enzyme in 0.1
M
potassium phosphate buffer, pH 7.0, at 30 °C using the guaiacol
spectrophotometric method [21].
4700 Z. Lamplot et al. (Eur. J. Biochem. 271) Ó FEBS 2004

the color intensity was substantially decreased.
After separation of the enzyme protein by ultrafiltration,
the GPAO/DAPY reaction mixture exhibited a fl uorescence
emission spectrum with a m aximum 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, R
f
¼ 0.62);
DAPY itself showed R
f
¼ 0.33 in the same system. Staining
for t ertiary a mines ( Draggendorff’s reagent) was a lso
positive for the GPAO/DAPY reaction mixture (an orange
spot, R
f
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

sium phosphate buffer, pH 7.0, after adding
0.1
M
DAPY (1 m
M
final concentratio n) at
30 °C. Intervals between scans: 15 s, total
time: 10 min.
Ó FEBS 2004 DAPY inactivates plant amine oxidases (Eur. J. Biochem. 271) 4701
Replacing DMAB in Kov acs’ reag ent 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 wi th the reagent and pro vided a
spectrum with a single peak centered at 510 nm. DMAB-
reacted 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
to form the corresponding aminoaldehydes, which sponta-
neously cyclize to 1-pyrroline and 1-piperideine, respect-
ively. The latter cyclic imines condense with ABA to
generate the corresponding substituted dihydroquinazolin-
ium compounds. The DAPY oxidation product obtained
by the reaction of GPAO provided an adduct with ABA

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 m
M
potassium phosphate buffer, pD 7.0 (made in D
2
Ofor
the purpose o f NMR spectroscopic analysis) r evealed
isotopic peaks belonging to quasimolecular ions of the
reaction product. The highest intensity was observed for
apeakwithm/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 a n instrumen t e quipped 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].
Fig. 4. Reaction of the DAPY oxidation product and a dihydropyridine

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 f ragmentation pattern
consistent wit h th e 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 m ass and elemental
composition of the DAPY oxidation product. An m/z
value of 178.14 was obtained, which matches a molecular
formula C
10
H
16
N
3
. Peaks in the corresponding MS/MS
spectrum provided the following m/z values and elemental
composition of ions: 161.11 (C
10
H
13
N
2
), 149.11 (C
9

reaction mixtures. (Upper) DAPY (5 m
M
)in
0.1
M
ammonium bicarbonate, pH 7.8, was
mixedwithanexcessofGPAOandincubated
at 30 °C for 24 h. After removing protein by
ultrafiltration, the reaction mixture was ana-
lyzedbyESI-IT-MSasdescribedinMaterials
and methods. (Lower) A combined solution of
DAPYandACA(each5m
M
)in0.1
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 ana-
lyzedbyESI-IT-MSasdescribedinMaterials
and methods.
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 collision-
induced fragmentation of the parent ion belonging to the DAPY
oxidation product (m/z 178.1).
Ó FEBS 2004 DAPY inactivates plant amine oxidases (Eur. J. Biochem. 271) 4703

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.
13
C-NMR spectra measured with the GPAO/
DAPY mixture in CD
3
OD resembled those recorded in
D
2
O, but the obtained quality was lower.
1
H-NMR spectra
were also recorded in D
2
O 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).
Other analyses
Lysine residues in plant CAOs are possible targets for
covalent binding of reactive electrophilic aminoaldehydes
formed by the turnover of acetylenic diamine substrates
[7–11]. The cDNA of GPAO subunit (without the signal

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.
Discussion
DAPY was synthesized as an analog of cadaverine
(pentane-1,5-diamine), which is known as the best substrate
Fig. 7.
1
H-NMR spectrum of DAPY oxidation product. DAPY (5 m
M
)in2mLof20m
M
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 CD
3
OD. The
insets shows a detailed view of the v inylic doubl et signals belonging to 2,3-dihydropyridine.
4704 Z. Lamplot et al. (Eur. J. Biochem. 271) Ó FEBS 2004
of plant CAOs [4]. Contrary to naturally occurring diam-
ines, the DAPY molecule contains a triple bond at the
b-andc-positions from the two primary amine termini. The
oxidative conversion of the compound by GPAO and
OVAO was demonstrated by measuring the production of

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 mechanism-
based 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 accom-
panied by spectral changes. The typical absorption spec-
trum 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
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-3-
pentynal 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 amino-
pentynal 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,

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 t he presence of a n itrogenous
heterocycle, in addition to a f ree amino group. The
GPAO/DAPY reaction mixture exhibited a positive reac-
tion 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 r eagents at the a-position of the heterocycle
[8].
The rigidity of the triple bond in the presumed amino-
pentynal 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 ad ded 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
Fig. 8. Chromatofocusing of DAPY-inactivated GPAO. Chromatofo-
cusing 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

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 N
1
-andN
5
-
(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 (C
9
H
8
N
2
)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
N
1
- and not the N
5
-isomer, and a small peak at m/z 120.1
(C
7
H
8

ACA-trapped DAPY oxidation product, wherein ACA
rather than unoxidized DAPY would add to the initial
aminopentynal turnover product (see Fig. 9).
According t o the mechanism for formation of t he
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, t he i nactivation
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 t he analogous product, N-(pyrrole-3-yl)-1,4-
diamino-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 signifi-
cantly less efficien cy than does t he 4-amino-2-butynal

gylamine terminus or the homopropargylamine terminus.
Our finding that both derivatives act as inactivators o f
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 e nzymes generate significant
amounts of two possible aminopentynal turnover products.
Either 5-amino-3-pentynal (after tautomerization) or
5-amino-2-pentynal can condense w ith a molecule of
unoxidized DAPY to give an adduct capable of cyclization
to the same 4-amino-2,3-dihydropyridine, which is appar-
ently resistant to hydrolysis on account of extended
resonance stabilization. The structure of the final product,
most likely N
1
- rather than N
5
-(2,3-dihydropyridin-4-yl)-
1,5-diamino-2-pentyne, was supported by spectrophoto-
metric, 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). Nonethe-
less, furthe r s tudies are needed to ascertain the true
molecular nature of enzyme modification lead ing to inac-
tivation by DAPY. Although DAPY is not such a powerful

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