Tài liệu Báo cáo khóa học: Inactivation of copper-containing amine oxidases by turnover products doc

Inactivation of copper-containing amine oxidases by turnover
products
Paola Pietrangeli
1
, Stefania Nocera
1
, Rodolfo Federico
2
, Bruno Mondovı
`
1
and Laura Morpurgo
1
1
Department of Biochemical Sciences ‘A. Rossi Fanelli’ and C.N.R. Institute of Molecular Biology and Pathology,
University of Rome ‘La Sapienza’, Rome, Italy;
2
Department of Biology, 3rd University of Rome, Rome, Italy
For bovine serum amine oxidase, two different mechanisms
of substrate-induced inactivation have been proposed. One
consists of a slow oxidation by H
2
O
2
of a conserved residue
in the reduced enzyme after the fast turnover phase [Pietr-
angeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovı
`
,
B. & Morpurgo, L. (2000) Biochem. Biophys. Res. Commun.
267, 174–178] and the other of the oxidation by H

2
O
2
of the
dihydrobenzoxazole in equilibrium with the product Schiff
base, during the catalytic cycle [Lee, Y., Shepard, E., Smith,
J., Dooley, D.M. & Sayre, L.M. (2001) Biochemistry 40,
822–829]. To discriminate between the two mechanisms, the
inactivation was studied using Lathyrus cicera (red vetch-
ling) amine oxidase. This, in contrast to bovine serum amine
oxidase, formed the Cu
+
-semiquinolamine radical with a
characteristic UV-vis spectrum when oxygen was exhausted
by an excess of any tested amine in a closed cuvette. The
inactivation, lasting about 90 min, was simultaneous with
the radical decay and with the formation of a broad band
(shoulder) at 350 nm. No inactivation occurred when a
thousand-fold excess of amine was rapidly oxidized in an
L. cicera amine oxidase solution stirred in open air. Thus,
the inactivation is a slow reaction of the reduced enzyme with
H
2
O
2
, following the turnover phase. Catalase protected
L. cicera amine oxidase from inactivation. This effect was
substrate-dependent, varying from full protection (benzyl-
amine) to no protection (putrescine). In the absence of H
2

O
2
,
a specific inactivating reaction, without formation of the
350 nm band, was induced by some aldehydes, notably
putrescine. Some mechanisms of inactivation are proposed.
Keywords: copper amine oxidase; trihydroxyphenylalanine
quinone; inactivation; hydrogen peroxide; aldehydes.
Copper-containing amine oxidases [amine:oxygen oxido-
reductase (deaminating) (copper containing); E.C.1.4.3.6]
are ubiquitous enzymes that catalyze the oxidative deami-
nation of primary amines, transferring two electrons to
molecular oxygen in a ping-pong reaction producing H
2
O
2
,
aldehydes, and ammonium ions [1,2].
E
ox
þ R-CH
2
-NH
þ
3
! E
red
-NH
þ
3

þ R-CHO
E
red
-NH
þ
3
þ O
2
! E
ox
þ NH
þ
4
þ H
2
O
2
Together with copper, they contain an organic prosthetic
group reactive with semicarbazide, phenylhydrazine and
similar inhibitors of catalytic activity. This prosthetic group
was identified [3] as trihydroxyphenylalanine quinone
(TPQ), a post-translationally oxidized tyrosine residue [4].
It has been known for some time that copper amine
oxidases are inactivated by the turnover product, H
2
O
2
,as
the presence of catalase protects the enzyme from inactiva-
tion. This was described for the diamine oxidases from pea

seedling [5] and pig kidney [6], and for the bovine serum
amine oxidase (BSAO) [7]. In the latter case, it was not
possible to identify the modification induced by H
2
O
2
, but
the similarity of the behavior of several amine oxidases
suggested that it consists of the oxidation of a conserved
residue at the active site [7]. Tryptophan or metal-coordi-
nated histidine residues, oxidized by H
2
O
2
in copper- and
manganese-containing superoxide dismutases, were not
affected in BSAO [7]. A more recent report [8] ascribed
BSAO inactivation by benzylamines to a partitioning
reaction, occurring during the catalytic cycle, between
H
2
O mediated hydrolysis of the product Schiff base, and
H
2
O
2
mediated oxidation of dihydrobenzoxazole in equi-
librium with it, yielding aldehyde and benzoxazole, respect-
ively. Inactivation by aldehydes is well documented for
plant amine oxidases, such as lentil seedling amine oxidase

treated with stoichiometric amounts of tryptamine under
anaerobic conditions [9], or treated under turnover condi-
tions with haloamines, 1,2-diaminoethane and 1,3-diamino-
propane [10], and with the mechanism-based inhibitor,
2-butyne-1,4-diamine [11].
The focus of the present work was whether amine
oxidase inactivation was due to H
2
O
2
reacting with the
reduced protein after the turnover phase [7] or with
Correspondence to P. Pietrangeli, Department of Biochemical Sciences
‘A. Rossi Fanelli’, University ‘La Sapienza’, P. le A. Moro 5,
00185 Rome, Italy. Fax: + 39 06 4440062, Tel.: + 39 06 49910639,
E-mail:
Abbreviations: LCAO, Lathyrus cicera amine oxidase; BSAO, bovine
serum amine oxidase; TPQ, 2,4,5-trihydroxyphenylalanine quinone;
AGAO, Arthrobacter globiformis amine oxidase; HPAO, Hansenula
polymorpha amine oxidase; ECAO, Escherichia coli amine oxidase.
Enzyme: amine:oxygen oxidoreductase (deaminating) (EC 1.4.3.6).
Dedication: This paper is dedicated to the memory of Eraldo Antonini,
eminent biochemist, who died prematurely 20 years ago on March
19th, 1983.
(Received 18 September 2003, revised 6 November 2003,
accepted 10 November 2003)
Eur. J. Biochem. 271, 146–152 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03913.x
dihydrobenzoxazole produced by partitioning during the
catalytic cycle [8]. The copper-containing amine oxidase
purified from Lathyrus cicera (red vetchling) seedling

(LCAO)wasusedasitformsCu
2+
-quinolamine in
equilibrium with Cu
+
-semiquinolamine under reducing
conditions as do other plant amine oxidases [2]. The
peculiar spectroscopic properties of the latter radical
allowed the inactivation process to be followed, while
BSAO was spectroscopically silent under similar conditions.
The first hypothesis [7] received supporting evidence and in
addition LCAO could also be inactivated by the aldehyde
produced by some substrates in the absence of H
2
O
2
.
Materials and methods
Protein purification
LCAO was purified from L. cicera seedlings using a simple
procedure that utilizes only two chromatographic steps
(Table 1). Seeds, obtained from the local market, were
soaked in aerated tap water for 12 h and grown in moistened
vermiculite for 7 days in the dark at 23 °C. Seedling shoots
(400 g) were homogenized in a Waring blender with 4 vols of
50 m
M
KH
2
PO

4
, pH 4.3. At this pH, most of the amine
oxidase activity (> 90%) associated with cell walls and fibres
was solubilized by increasing the ionic strength. The homo-
genate was then strained through cheese cloth and the solid
residue was washed four times with 3 vols of the same buffer.
The enzyme was then eluted with 1 vol of 20% saturated
ammonium sulphate in 50 m
M
KH
2
PO
4
,pH4.3.The
suspension was pressed through cheese cloth, and centri-
fuged at 15 000 g for 30 min. The supernatant was brought
to pH 7.0 with KOH, and to 70% saturation with solid
ammonium sulphate, then it was stirred for 30 min and
centrifuged at 15 000 g for 30 min. Although the purification
was less than twofold, the advantage of this procedure was a
substantial volume reduction. The precipitate was collected,
resuspended in 0.2 vols of 15 m
M
potassium phosphate
buffer, pH 7 and dialyzed overnight against the same buffer.
The dialysate was loaded onto a DEAE–cellulose (What-
man) column (20 · 4 cm i.d.) equilibrated with 15 m
M
potassium phosphate pH 7.0. During loading and washing
with buffer, the eluate at A

280
> 0.1 was collected. As the
enzyme did not bind, the solution, containing more than
80% of the total loaded amine oxidase activity, was adjusted
to pH 5.5 with 1
M
H
3
PO
4
and applied directly onto a SP
Hi Trap (Pharmacia) column (5 · 0.8 cm i.d.) equilibrated
with 50 m
M
potassium phosphate buffer, pH 5.5. The
column was washed with the same buffer and also with the
same buffer containing 0.1
M
NaCl, then the amine oxidase
was eluted using buffer containing 0.2
M
NaCl. Fractions
with high enzymatic activity were pooled and analyzed.
Activity and protein assays
The purified proteins moved as single bands on SDS/
PAGE. The concentration was measured by employing the
molar extinction coefficients reported for the pea seedling
enzyme (PSAO) [12], namely e
280nm
¼ 300 000

M
)1
Æcm
)1
and e
500nm
¼ 4900
M
)1
Æcm
)1
(Results). The copper content
was assayed by atomic absorption spectrometry with a
Perkin Elmer apparatus equipped with a HGA-400
graphite furnace and by the biquinoline spectrophotomet-
ric method [13]. The amine oxidase activity was assayed
spectrophotometrically at 25 °C with 1.0 m
M
putrescine in
0.1
M
potassium phosphate buffer, pH 7.2, by measuring
the formation of H
2
O
2
from the absorbance of the pink
adduct (e
515nm
¼ 2.6 · 10

4
M
)1
Æcm
)1
) produced by the
horseradish peroxidase catalyzed oxidation of aminoanti-
pyrine, followed by condensation with 3,5-dichloro-2-
hydroxybenzensulfonic acid [14]. Samples with specific
activity ¼ 70 IUÆmg
)1
(micromoles of substrate oxid-
izedÆper min) were employed. The vis-UV spectra were
recorded with an AVIV (Lakewood, NJ, USA) spectro-
photometer, Model 14 DS, equipped with a thermostatted
cell holder.
Chemicals
Amines, aminoantipyrine, 3,5-dichloro-2-hydroxybenzen-
sulfonic acid, catalase, horse radish peroxidase were
purchased from Sigma Chemical Co. All other chemicals
were commercial products of analytical purity grade.
Steady-state kinetic measurements
Kinetic data were obtained by measuring the velocity of
H
2
O
2
formation as described above for the enzymatic
activity determination. K
m

and k
cat
values were obtained by
fitting the kinetic data to the Michaelis–Menten equation
v ¼ V
max
[S]/(K
m
+ [S]) by nonlinear regression analysis
using Microcal
ORIGIN
3.5 software. The data were the
average of two/three experiments, carried out at 25 °C,
using at least eight amine concentrations each. The standard
error was ± 8%. The catalytic parameters were measured in
0.1
M
potassium phosphate buffer, pH 7.2, at 120 m
M
ionic
strength. Tris/HCl buffer at 0.1
M
, pH 7.2 and 120 m
M
ionic strength was used for spermine.
LCAO inactivation by amines
The experiments were performed under the same conditions
as described previously for BSAO [7], that is by incubating
0.4 l
M

LCAO with substrate, in 0.1
M
potassium phosphate
buffer, at three different pH values of 6.5, 7.2 and 8.0 in a
Table 1. Purification of LCAO.
Purification step
Total Volume
(mL)
Total activity
(IU)
Total protein
(mg)
Specific activity
(IUÆmg
)1
)
Purification
(fold)
Yield
(%)
Crude extract 440 2540 190 13.4 1 100
(NH
4
)
2
SO
4
70% precipitate, dialysis 65 1790 80 22.5 1.7 70
DEAE–cellulose chromatography 70 1420 45 32 2.5 57
SP Hi Trap chromatography 7 1260 18 70 5.2 50

Ó FEBS 2003 Inactivation of copper-containing amine oxidases (Eur. J. Biochem. 271) 147
1 mL test tube, open to air, using a 37 °C thermostatted
water bath. At given time intervals, aliquots of the solutions
were tested for activity with 1.0 m
M
putrescine at 25 °C,
after dilution to approximately 2 n
M
LCAO. In another set
of experiments the inactivation was carried out by incuba-
ting LCAO (4.0–6.0 l
M
) with 1.0 m
M
substrate, both at
25 °Cand37°C, in a cuvette provided with a Teflon
stopper, thus limiting the amount of available oxygen and
allowing the monitoring of the UV-vis spectrum of reacting
species.
Results
LCAO purification and characterization
The purification method described above was more rapid
and allowed a larger recovery than the previously reported
one [15]. Thus, it is highly suitable for a large scale
preparation of LCAO.
Molecular and enzymatic properties of LCAO were
found to be very similar to those of other Cu-containing
amine oxidases, particularly those from plant sources. As
reported in Materials and methods, the LCAO concentra-
tion was measured by using the PSAO molar extinction

coefficients [12]. These values were chosen because they
provided protein concentrations, identical at 280 nm and
500 nm, in good agreement with the copper content of
2.0 ± 0.1 ions per dimer and with the content of reactive
TPQ groups. Lower coefficients were reported previously
for LCAO [15] and for the homologous enzyme from
Lathyrus sativus [16]. The content of TPQ was measured by
titration with benzylhydrazine and with 2-hydrazinopyri-
dine. The reaction with benzylhydrazine produced a stable
adduct absorbing at 380 nm, with an extinction coefficient
e
380nm
¼ 65 000
M
)1
Æcm
)1
, accounting for 1.9 ± 0.1 TPQ
per dimer. These properties were quite similar to those of
the corresponding adducts of LSAO [17] and PSAO [12].
The reaction with 2-hydrazinopyridine formed an adduct
absorbing at 420 nm, with e
420nm
¼ 58 000
M
)1
Æcm
)1
,
accounting for 1.8 ± 0.1 TPQ groups per dimer.

LCAO steady-state kinetic parameters
Table 2 reports the steady-state kinetic parameters of the
LCAO catalyzed oxidation of some primary amines. All
kinetic measurements were carried out on protein samples
from the same batch. The substrates are arranged in the
table in order of decreasing k
cat
, which shows  500-fold
decrease in the list. The values of k
cat
/K
m
are less variable
with the exception of putrescine and cadaverine.
LCAO inactivation in air
LCAO was irreversibly inactivated, as is BSAO, by
incubation with excess amine. Table 3 shows the residual
activity after 30 and 90 min incubation with some substrates
in a test tube opened to air at 37 °C. The loss of activity was
dependent upon the incubation time and the amine
concentration and much less on the nature of the amine
used. In general it approached a value of 60% after 30 min,
and of > 90% after 90 min.
Inactivation was reduced to about 30% after 20 and
60 min using gentle shacking in a water bath and
abolished by very efficient stirring. The latter result was
achieved by introducing 300 lL of a solution that
contained 2.0 l
M
LCAO and 2.0 m

M
cadaverine, in a
3 mL spectrophotometer cuvette provided with a small
magnetic stirrer and thermostatted at 25 °C. Aliquots of
the solution were withdrawn at time intervals and tested
for activity and H
2
O
2
content, after proper dilution. No
loss of activity occurred in these conditions after 2, 5 and
20 min, although all of the amine was already oxidized
after 2.0 min, as measured by the concentration of
produced H
2
O
2
. Stirred LCAO was neither inactivated
during the turnover phase, nor in the phase subsequent to
amine exhaustion.
The inactivation was also reduced by the presence of
catalase, especially in the first 30 min. The effect of catalase
was considerably dependent on the substrate, varying from
full protection with benzylamine and agmatine to no
protection at all with putrescine. Changes of pH in the
range 6.5–8.0 had a relatively small effect on the inactiva-
tion, while the protecting effect of catalase was usually
larger at pH 6.5 than at pH 8.0. Figure 1 shows the results
obtained with spermidine at three different pH values as an
example.

UV-vis effects of LCAO inactivation
As described in Materials and methods, the process of
inactivation was monitored spectrophotometrically by
incubating LCAO (4.0–6.0 l
M
)at37°Cor25°C, with
1.0 m
M
substrate in a spectrophotometer cuvette closed by
a Teflon stopper. The oxygen was consumed by most
substrates within the mixing time as revealed by the
immediate bleaching of the TPQ 500 nm band and by the
appearance of absorption peaks at 465, 435 and 360 nm,
identical with those of the Cu
+
-semiquinolamine radical,
which is formed under anaerobic conditions by some
copper amine oxidases [2]. With some substrates, such as
2-aminomethylpyridine, a few minutes were required to
develop the radical signal, that is to exhaust the oxygen,
but this did not change the general behavior. In any case,
the spectrum of the radical showed a similar intensity at a
given temperature and faded slowly away within about
90 min, together with the catalytic activity and with the
formation of a broad band (shoulder) at  350 nm. At
25 °C, the initial intensity of the spectrum was lower than
Table 2. Steady state kinetics: k
cat
and substrate specificity (k
cat

/K
m
)
for the oxidative deamination of primary amines catalyzed by LCAO.
Substrate k
cat
(s
)1
) k
cat
/K
m
(s
)1
Æ
M
)1
)
Putrescine 262 0.97 · 10
6
Cadaverine 159 1.6 · 10
6
Spermidine 100 4.8 · 10
4
Agmatine 45.9 0.94 · 10
5
Tyramine 32.9 1.1 · 10
4
Spermine 28.3 4.5 · 10
4

Histamine 10.3 1.3 · 10
4
Benzylamine 3.7 0.90 · 10
4
2-Aminomethylpyridine 0.47 1.5 · 10
4
148 P. Pietrangeli et al. (Eur. J. Biochem. 271) Ó FEBS 2003
at 37 °C, in agreement with the reported temperature
dependence of the equilibrium between Cu
+
-semiquol-
amine and Cu
2+
-quinolamine [18]. The inactivation was
also slower at 25 °Cthanat37°C, but the overall
behavior was similar. No recovery of activity or spectro-
scopic properties occurred after extensive dialysis. The
addition of 2-hydrazinopyridine up to a concentration of
0.1 m
M
, to solutions that were not completely inactivated,
slowly formed a band at 420 nm, typical of the 2-hydraz-
inopyridine adduct of TPQ. The final band intensity
matched the residual activity of the solution. The reaction
was slow, implying previous reoxidation of active mole-
cules by oxygen and competition of 2-hydrazinopyridine
with excess substrate. Also implied is that the TPQ of
inactivated molecules was no longer able to bind
inhibitors.
The decay of the radical spectrum with time, which was

very similar with all substrates, is shown in Fig. 2 for
putrescine. This substrate was chosen because of its different
behavior from other substrates in the presence of catalase
(Table 2 and below). Isosbestic points are present in Fig. 2,
at least in the early stages of the reaction, because of the
broad band (shoulder) formed in the 350 nm region. By
subtracting the spectrum of native LCAO from the
spectrum of the radical, or from the spectra of the
inactivated protein, a peak around 310–320 nm was
observed with all substrates. The peaks produced by
putrescine are shown in Fig. 3. The band at 350 nm is
Table 3. Residual LCAO activity after incubation with substrate. Experimental conditions: 2.8 l
M
LCAO, 2 m
M
substrate, unless otherwise stated;
0.1
M
potassium phosphate buffer pH 7.2, 37 °C. The activity was measured at 25 °C after proper sample dilution.
Substrate
[Amine]
(m
M
)
Residual activity
(% of starting activity)
Residual activity in the
presence of catalase
(% of starting activity)
Time (min) Time (min)

30 90 30 90
Putrescine 2.0 0 0 0 0
0.5 40 20 42 25
Cadaverine 2.0 20 17 79 77
10.0 3.0 3.0 75 48
Spermidine 2.0 19 6.0 89 43
Agmatine 2.0 41 37 100 100
Tyramine 2.0 10 10 70 70
Spermine 2.0 44 10 85 15
10.0 15 2.0 51 8.0
Histamine 2.0 35 30 90 90
Benzylamine 2.0 40 30 100 100
2-Aminomethylpyridine 2.0 45 15 55 35
Fig. 1. Time course of LCAO inactivation by spermidine. LCAO,
2.8 l
M
, was inactivated at 37 °Cby2.0m
M
substrate in 0.1
M
potas-
sium phosphate buffer at different pH values (open symbols) and in
presence of catalase (full symbols): pH 6.5 (triangles); pH 7.2 (circles);
pH 8.0 (squares).
Fig. 2. Decrease with time of the UV-vis spectrum of the radical formed
by putrescine-reacted LCAO. The spectra were recorded 1 (top spec-
trum), 10, 30, 50, 70, 90, 120 min after the addition of 1.0 m
M
putrescine to 5.5 l
M

LCAO, in 0.1
M
potassium phosphate buffer
pH 7.2, at 37 °C.
Ó FEBS 2003 Inactivation of copper-containing amine oxidases (Eur. J. Biochem. 271) 149
evident in the spectrum of the inactivated protein. A peak at
315 nm was found in the difference spectra of inactivated
BSAO and was taken to be diagnostic of reduced TPQ
[7,19]. The slight variability of the peak maximum wave-
length may be due to the fact that this is not a real band but
the result of the bleaching of an intense TPQ band at
270 nm [20].
In an inactivation experiment carried out at 25 °C, in
order to slow down the reaction to obtain more accurate
readings, the residual activity was measured in solution
aliquots withdrawn after recording the intensity of the
465 nm peak. Five minutes after substrate addition, the
radical was formed and the protein was fully active. Then
the loss of activity and the loss of radical intensity took place
at almost coincident rates (Fig. 4). At the end of the
experiment, a concentration of 0.25 ± 0.01 m
M
H
2
O
2
was
measured immediately after the cuvette was opened to air, in
good agreement with the initial oxygen content of the
solution, indicating that significant oxygen leaks had not

occurred during the incubation and that a similar amount of
oxygen was available in all experiments performed at the
same temperature. This was confirmed by incubating
0.3 l
M
LCAO with 1 m
M
putrescine, at 25 °C, in a 2-mm
path-length cuvette opened to air, which also contained the
H
2
O
2
-detecting system. The absorbance at 515 nm reached
a value of 1.27 (corresponding to 0.245 m
M
H
2
O
2
)within
5 min, remained constant for about 15 min, then started to
decrease. The decrease was of 2% in the subsequent 40 min,
while a dark red layer about 2 mm thick formed at the
surface of the solution. This experiment demonstrated that
in the absence of stirring, extra oxygen is not readily
available in the bulk solution after exhaustion of the initial
amount present and that diffusion is prevented by the
reaction with excess amine in the top layer. In stirred
solutions, all the amine was oxidized within 2 min, as shown

by the equivalent amount of H
2
O
2
detected in solution at
this stage. Thus, the turnover of a thousand-fold amine
excess did not cause inactivation as the protein remained
reduced for a too short time.
Inthepresenceofcatalase,thedecayoftheradical
spectrum either did not occur, as in the case of cadaverine,
in agreement with the results on PSAO [5], or was greatly
reduced, to  20% in the case of spermine (not shown). The
only exception was putrescine (Fig. 5). The decay of the
radical, slower than in absence of catalase, did not produce
isosbestic points nor a shoulder in the 350 nm region, while
the 500 nm band of the cofactor remained bleached.
Discussion
The process of BSAO inactivation required a long
incubation with substrate, was inhibited by the presence
of catalase, which eliminates H
2
O
2
, but was not produced
by exogenous H
2
O
2
added to the resting enzyme [7].
These results were taken to imply that the inactivation is a

Fig. 3. Difference spectra of putrescine-reacted LCAO. LCAO, 7.8 l
M
,
was reacted with 1.0 m
M
putrescine. The spectra, recorded immedi-
ately (solid line) and after 5 h incubation (dashed line) were subtracted
of the native protein spectrum. 0.1
M
potassium phosphate buffer
pH 7.2 at 25 °C.
Fig. 4. LCAO inactivation. Decay with time of the enzyme activity (s)
and of the radical band at 465 nm (d) upon addition of 1.0 m
M
putrescine to 4.4 l
M
LCAO in 0.1
M
potassium phosphate buffer
pH 7.2 at 25 °C.Theactivitybeforeputrescineadditionwastakenas
100.
Fig. 5. Decrease with time of the UV-vis spectrum of the radical formed
by putrescine-reacted LCAO in the presence of catalase. The spectra
were recorded 1 (top spectrum), 10, 40, 70, 110, 150 min after addition
of 1.0 m
M
putrescine to 4.4 l
M
LCAO, in 0.1
M

potassium phosphate
buffer pH 7.2, at 37 °C, in the presence of 100 catalase units.
150 P. Pietrangeli et al. (Eur. J. Biochem. 271) Ó FEBS 2003
slow process, involving H
2
O
2
and a substrate-reduced
form of the protein. These conclusions are confirmed by
the similar results obtained with LCAO. The alternative
mechanism proposed involving a partitioning reaction
during turnover [8] was excluded as LCAO was fully
active after either oxygen was consumed by an excess of
amine in a closed cuvette, or the amine was consumed by
oxygen in a solution stirred in air, with the rapid turnover
of a thousand-fold amine excess. Furthermore, the loss of
activity in a closed cuvette was a slow reaction, subse-
quent to oxygen exhaustion and the turnover phase,
simultaneous with the loss of intensity of the UV-vis
spectrum of the Cu
+
-semiquinolamine radical (Fig. 4). All
substrates displayed a similar inactivation time, independ-
ent of their highly different catalytic parameters (Table 2)
and formed the same band around 350 nm in a closed
cuvette. This shows that the reaction was independent of
the substrate or related aldehyde and that H
2
O
2

, radical
and/or Cu
2+
-quinolamine were the only reacting species.
Each species was always present at same concentration, as
shown by the measured amounts of H
2
O
2
, by the initial
intensity of the radical spectrum and by the bleached
500 nm band. Which of the two equilibrium species,
the radical or the Cu
2+
-quinolamine, participated in the
reaction is not certain. On one hand, the decay of the
radical to the broad band at 350 nm formed isosbestic
points along a large part of the reaction [Fig. 3]. On the
other hand, BSAO was similarly inactivated [7], although
it does not form the radical [2].
A possible explanation of these results is suggested by a
recent report [21], in which the quinolamine was prepared
by reducing Co- and Ni-substituted Arthrobacter globi-
formis amine oxidase (AGAO) with substoichiometric
amounts of substrate under anaerobic conditions. By
addition of an excess of exogenous aldehyde, a band at
350 nm was slowly formed, which was assigned to a back-
reaction generating the neutral form of the product Schiff
base. This is more stable than the protonated form [22]
preferred by Cu-AGAO, toward hydrolysis to aldehyde and

quinolamine. The band disappeared on admission of
oxygen into the solution. The band at 350 nm formed by
LCAO, upon bleaching of the radical, suggests that the
neutral form of the product Schiff base was stabilized by the
modification responsible for the loss of catalytic activity,
causing back-reaction of aldehydes with Cu
2+
-quinol-
amine. This implies that the reaction with H
2
O
2
did not
modify the quinolamine but oxidized another conserved
residue at the active site. The similar inactivation time of all
substrates suggests that the oxidation by H
2
O
2
was the rate-
determining step, slower than the back-reaction with
aldehyde. At difference from the 350 nm band formed by
Co- and Ni-AGAO, the LCAO band was not affected by
the admission of oxygen in solution. Thus, the inactivated
protein was unable to hydrolyze the aldehyde and to react
with oxygen. In previous work on BSAO [7] it was proposed
that the H
2
O
2

target is a conserved residue affected by TPQ
reduction. This residue was tentatively identified as Tyr371,
corresponding to Tyr369 in E. coli amine oxidase [23,24] or
Tyr305 in Hansenula polymorpha amine oxidase [25]. The
short hydrogen bond (2.4 A
˚
)ofTyr369toTPQO4in
ECAO has been taken to imply O4 deprotonation [23]. The
TPQ O4 basic character increases considerably in the
reduced cofactor [20], causing a partial deprotonation of the
Tyr hydroxyl. The mutation of Tyr305 in HPAO [25] and
Tyr369 in ECAO [24] decreased the enzyme catalytic
activity, to a variable degree depending on the type of
mutation and modified the active site hydrogen bond
network and cofactor mobility.
In the presence of catalase, some substrates produced
partial inactivation when high aldehyde concentrations were
reached upon prolonged incubation in air (Table 2). The
effect does not appear to be related to the kinetic and
structural properties of the substrate but rather to the
specific reactivity of the corresponding aldehyde. This is
evident in the benzylamine/agmatine or putrescine/cadaver-
ine couples. The behavior of putrescine was unique as it was
able to inactivate completely the protein in absence of H
2
O
2
.
The process bleached the radical spectrum without forma-
tion of the 350 nm band [Fig. 5]. The back-reaction of the

aldehyde or pyrroline with quinolamine did not occur, as
the neutral form of the product Schiff base was not
stabilized in absence of H
2
O
2
. The aldehyde or pyrroline
may react with a nucleophilic residue as often reported for
plant amine oxidases [9–11]. BSAO has a very low reactivity
with this substrate, k
cat
¼ 0.017 s
)1
[26].
In conclusion, the inactivation is a slow reaction of the
reduced protein with H
2
O
2
, subsequent to turnover and
occurring in a similar way for all amines examined. As it is
common to all copper amine oxidases investigated so far, it
might be relevant to some in vivo functions of amine
oxidases.
Acknowledgements
This paper was supported by Murst and by a C.N.R. grant no.
G002FD1 Agenzia 2000.
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