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 i
Trang 1Inactivation of copper-containing amine oxidases by turnover
products
Paola Pietrangeli1, Stefania Nocera1, Rodolfo Federico2, Bruno Mondovı`1and Laura Morpurgo1
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;2Department 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 H2O2of 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 H2O2of 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 ciceraamine oxidase solution stirred in open air Thus, the inactivation is a slow reaction of the reduced enzyme with
H2O2, following the turnover phase Catalase protected
L ciceraamine oxidase from inactivation This effect was substrate-dependent, varying from full protection (benzyl-amine) to no protection (putrescine) In the absence of H2O2,
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 H2O2,
aldehydes, and ammonium ions [1,2]
Eoxþ R-CH2-NHþ3 ! Ered-NHþ3 þ R-CHO
Ered-NHþ3 þ O2! Eoxþ NHþ
4 þ H2O2 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, H2O2, 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 H2O2, 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 H2O2in 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
H2O mediated hydrolysis of the product Schiff base, and
H2O2 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 H2O2 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: paola.pietrangeli@uniroma1.it
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)
Trang 2dihydrobenzoxazole produced by partitioning during the
catalytic cycle [8] The copper-containing amine oxidase
purified from Lathyrus cicera (red vetchling) seedling
(LCAO) was used as it forms Cu2+-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 H2O2
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 23C Seedling shoots
(400 g) were homogenized in a Waring blender with 4 vols of
50 mM KH2PO4, 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 mM KH2PO4, pH 4.3 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 mM 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 mM
potassium phosphate pH 7.0 During loading and washing
with buffer, the eluate at A280> 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 1MH3PO4and applied directly onto a SP
Hi Trap (Pharmacia) column (5· 0.8 cm i.d.) equilibrated
with 50 mM potassium phosphate buffer, pH 5.5 The
column was washed with the same buffer and also with the
same buffer containing 0.1MNaCl, then the amine oxidase
was eluted using buffer containing 0.2MNaCl 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 e280nm¼ 300 000M )1Æcm)1 and e500nm¼ 4900M )1Æcm)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 25C with 1.0 mMputrescine in 0.1M potassium phosphate buffer, pH 7.2, by measuring the formation of H2O2 from the absorbance of the pink adduct (e515nm¼ 2.6 · 104M )1Æcm)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 activity¼ 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
H2O2 formation as described above for the enzymatic activity determination Kmand kcatvalues were obtained by fitting the kinetic data to the Michaelis–Menten equation
v¼ Vmax [S]/(Km+ [S]) by nonlinear regression analysis using Microcal ORIGIN 3.5 software The data were the average of two/three experiments, carried out at 25C, using at least eight amine concentrations each The standard error was ± 8% The catalytic parameters were measured in 0.1Mpotassium phosphate buffer, pH 7.2, at 120 mMionic strength Tris/HCl buffer at 0.1M, pH 7.2 and 120 mM 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 lMLCAO with substrate, in 0.1Mpotassium 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 (%)
(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
Trang 31 mL test tube, open to air, using a 37C thermostatted
water bath At given time intervals, aliquots of the solutions
were tested for activity with 1.0 mM putrescine at 25C,
after dilution to approximately 2 nMLCAO In another set
of experiments the inactivation was carried out by
incuba-ting LCAO (4.0–6.0 lM) with 1.0 mM substrate, both at
25C and 37 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 extincconcentra-tion
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
e380nm¼ 65 000M )1Æcm)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 e420nm¼ 58 000M )1Æcm)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 kcat, which shows 500-fold decrease in the list The values of kcat/Kmare 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 37C 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 lM LCAO and 2.0 mM cadaverine, in a
3 mL spectrophotometer cuvette provided with a small magnetic stirrer and thermostatted at 25C Aliquots of the solution were withdrawn at time intervals and tested for activity and H2O2 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 H2O2 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 lM) at 37C or 25 C, with 1.0 mMsubstrate 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
25C, 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)1Æ 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
Trang 4at 37C, in agreement with the reported temperature
dependence of the equilibrium between Cu+
-semiquol-amine and Cu2+-quinolamine [18] The inactivation was
also slower at 25C than at 37 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 mM, 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)
Fig 1 Time course of LCAO inactivation by spermidine LCAO,
2.8 l M , was inactivated at 37 C by 2.0 m 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.
Trang 5evident 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 25C, 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 mMH2O2was
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 lMLCAO with 1 mMputrescine, at 25C, in a 2-mm
path-length cuvette opened to air, which also contained the
H2O2-detecting system The absorbance at 515 nm reached
a value of 1.27 (corresponding to 0.245 mMH2O2) 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 H2O2detected 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
In the presence of catalase, the decay of the radical 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 H2O2, but was not produced
by exogenous H2O2 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 The activity before putrescine addition was taken as 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.
Trang 6slow process, involving H2O2 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 differindepend-ent 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 H2O2, radical
and/or Cu2+-quinolamine were the only reacting species
Each species was always present at same concentration, as
shown by the measured amounts of H2O2, 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 Cu2+-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 Cu2+
-quinol-amine This implies that the reaction with H2O2 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 H2O2was 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 H2O2target 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˚) of Tyr369 to TPQ O4 in
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 putrescputrescine/cadaver-ine was unique as it was able to inactivate completely the protein in absence of H2O2 The process bleached the radical spectrum without forma-tion of the 350 nm band [Fig 5] The back-reacforma-tion 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 H2O2 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, kcat¼ 0.017 s)1[26]
In conclusion, the inactivation is a slow reaction of the reduced protein with H2O2, 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 (Davidson, V.L., ed.), pp 97–171 Marcel Decker, New York.
2 Padiglia, A., Medda, R., Bellelli, A., Agostinelli, E., Morpurgo, L., Mondovı`, B., Finazzi Agro`, A & Floris, G (2001) The reductive and oxidative half-reactions and the role of copper-ions in plant and mammalian copper-amine oxidases Eur J Inorg Chem 35–42.
3 Janes, S.M., Mu, D., Wemmer, D., Smith, A.J., Kaur, S., Maltby, D., Burligame, A.L & Klinman, J.P (1990) A new redox cofactor
in eucariotic enzymes: 6-hydroxy-dopa at the active site of bovine serum amine oxidase Science 248, 981–987.
4 Cai, D & Klinman, J.P (1994) Evidence for a self-catalytic mechanism of 2,4,5-trihydroxyphenylalanine quinone biogenesis
in yeast copper amine oxidase J Biol Chem 269, 32039–32042.
5 Mann, P.J.G (1955) Purification and properties of the amine oxidase of pea seedlings Biochem J 59, 609–620.
6 Mondovı`, B., Rotilio, G., Finazzi-Agro`, A & Costa, M.T (1967) Diamine oxidase inactivation by hydrogen peroxide Biochim Biophys Acta 132, 521–523.
7 Pietrangeli, P., Nocera, S., Fattibene, P., Wang, X.T., Mondovı`.
B & Morpurgo, L (2000) Modulation of bovine serum amine 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 O -dependent
Trang 7inactivation: 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., Sˇebela, 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-based inhibitor 2-butyne-1,4-diamine Eur J Biochem 267,
1423–1433.
12 McGuirl, M.A., McCahon, C.D., McKeown, K.A & Dooley,
D.M (1994) Purification and characterization of pea seedling
amine oxidase for crystallization studies Plant Physiol 106,
1205–1211.
13 Brumby, P.E & Massey, V (1967) Determination of nonheme
iron, total iron and copper Methods Enzymol 10, 463–474.
14 Angelini, R., Rea, G., Federico, R & D’Ovidio, R (1996) Spatial
distribution and temporal accumulation of mRNA encoding
diamine oxidase during lentil (Lens culinaris Medicus) seedling
development Plant Sci 119, 103–113.
15 Cogoni, A., Farci, L., Medda, R., Rinaldi, A & Floris, G (1989)
Amine oxidase from Lathyrus cicera and Phaseolus vulgaris:
puri-fication and properties Preport Biochem 19, 95–112.
16 Sˇebela, M., Luhova, L., Fre´bort, I., Fullhammer, H., Hirota, S.,
Zajonkova, L., Stuzka, V & Pec, P (1998) Analysis of the active
sites of the copper/topa quinone-containing amine oxidases from
Lathyrus odoratus and L sativus seedlings Phytochem Anal 9,
211–222.
17 Padiglia, A., Medda, R & Floris, G (1992) Lentil seedling amine
oxidase: interaction with carbonyl reagents Biochem Int 28,
1097–1107.
18 Dooley, D.M., McGuirl, M.A., Brown, D.E., Turowsky, P.N.,
McIntire, W.S & Knowles, P.F (1991) A Cu(I)-semiquinone state
in substrate-reduced amine oxidases Nature 349, 262–264.
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) Role of 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 the quinoenzyme amine oxidase from Escherichia 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 cofactor biogenesis in a copper amine oxidase Biochemistry 38, 3683–3693.
26 De Matteis, G., Agostinelli, E., Mondovı`, B & Morpurgo, L (1999) The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors J Biol Inorg Chem 4, 348–353.