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

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

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

(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

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

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

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

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.

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.

/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

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)

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

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

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

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

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.
References
1. McIntire, W.S. & Hartmann, C. (1992) Copper containing amine
oxidases. In Principles and Applications of Quinoproteins

oxidase activity by hydrogen peroxide. Biochem. Biophys. Res.
Commun. 267, 174–178.
8. Lee, Y., Shepard, E., Smith, J., Dooley, D.M. & Sayre, L.M.
(2001) Catalytic turnover of substrate benzylamines by the qui-
none-dependent plasma amine oxidase leads to H
2
O
2
-dependent
Ó FEBS 2003 Inactivation of copper-containing amine oxidases (Eur. J. Biochem. 271) 151
inactivation: evidence for generation of a cofactor-derived ben-
zoxazole. Biochemistry 40, 822–829.
9. Medda, R., Padiglia, A., Finazzi-Agro
`
, A., Pedersen, J.Z., Lorrai,
A. & Floris, G. (1997) Tryptamine as substrate and inhibitor of
lentil seedling copper amine oxidase. Eur. J. Biochem. 250, 377–382.
10. Medda, R., Padiglia, A., Pedersen, J.Z., Finazzi- Agro
`
,A.,
Rotilio, G. & Floris, G. (1997) Inhibition of copper amine oxidase
by haloamines. A killer product mechanism. Biochemistry 36,
2595–2602.
11. Fre
´
bort, I., S
ˇ
ebela, M., Svendsen, I., Hirota, S., Masaaki, E.,
Yamauchi, O., Bellelli, A., Lemr, K. & Pec, P. (2000) Molecular
mode of interaction of plant amine oxidase with the mechanism-

19. Su, Q. & Klinman, J.P. (1998) Probing the mechanism of proton
coupled electron transfer to dioxygen: the oxidative half-
reaction of bovine serum amine oxidase. Biochemistry 37, 12513–
12525.
20. Bossa, M., Morpurgo, G.O. & Morpurgo, L. (1994) Models and
molecular orbital semiempirical calculations in the study of the
spectroscopic properties of bovine serum amine oxidase quinone
cofactor. Biochemistry 33, 4425–4431.
21. Kishishita, S., Okajima, T., Kim, M., Yamaguchi, H., Hirota, S.,
Suzuki,S.,Kuroda,S.,Tanizawa,K.&Mure,M.(2003)Roleof
copper ion in bacterial copper amine oxidase: spectroscopic and
crystallographic studies of metal-substituted enzymes. J. Am.
Chem. Soc. 125, 1041–1055.
22. Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J. & Klinman,
J.P. (1997) Mechanism-based inactivation of a yeast methylamine
oxidase mutant: Implications for a functional role of the consensus
sequence surrounding topaquinone. Biochemistry 36, 11472–
11478.
23. Wilmot, C.M., Murray, J.M., Alton, G., Parsons, M.R., Convery,
M.A., Blakeley, V., Corner, A.S., Palcic, M.M., Knowles, P.F.,
McPherson, M.J. & Phillips, S.E.V. (1997) Catalytic mechanism of
thequinoenzymeamineoxidasefromEscherichia coli:exploring
the reductive half-reaction. Biochemistry 36, 1608–1620.
24. Murray, J.M., Kurtis, C.R., Tambyrajah, W., Saysell, C.G.,
Wilmot, C.M., Parsons, M.R., Phillips, S.E.V., Knowles, P.F. &
McPherson, M.J. (2001) Conserved tyrosine-369 in the active
site of Escherichia coli copper amine oxidase is not essential.
Biochemistry 40, 12808–12818.
25. Hevel, J.M., Mills, S.A. & Klinman, J.P. (1999) Mutation of a
strictly conserved, active-site residue alters substrate specificity and