Báo cáo khoa học: Active site residue involvement in monoamine or diamine oxidation catalysed by pea seedling amine oxidase doc

Active site residue involvement in monoamine or diamine
oxidation catalysed by pea seedling amine oxidase
Maria Luisa Di Paolo
1
, Michele Lunelli
2
, Monika Fuxreiter
2,3
, Adelio Rigo
1
, Istvan Simon
3
and Marina Scarpa
2
1 Dipartimento di Chimica Biologica and INBB, Universita
`
di Padova, Padova, Italy
2 Dipartimento di Fisica, Universita
`
di Trento, Trento, Italy
3 Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary
Introduction
Copper amine oxidases (CuAOs; EC 1.4.3.6) are wide-
spread in nature, being present in both prokaryotic
and eukaryotic organisms. They are homodimers, each
subunit containing a copper and a redox cofactor,
2,4,5-trihydroxyphenylalanine quinone (TPQ) [1].
CuAOs catalyse the oxidative deamination of primary
amines to the corresponding aldehydes, according to
the overall reaction:
RCH

2
NH
þ
3
þ O
2
þ H
2
O ! RCHO þ NH
þ
4
þ H
2
O
2
Catalysis occurs by a ping-pong mechanism, in which
the amine is converted to the product aldehyde while
reducing the enzyme cofactor (reductive half-reaction);
this is followed by reoxidation of the cofactor by
oxygen, which completes the catalytic cycle (oxidative
half-reaction) [2].
To date, several amine oxidase crystal structures
have been solved [3–10]. The structures for Escherichia
coli (ECAO) [3], Pisum sativum (PSAO) [4], Arthro-
bacter globiformis (AGAO) [5], Hansenula polimorpha
(HPAO) [6], Pichia pastoris [7], bovine serum amine
oxidase (BSAO) [8] and human semicarbazide sensitive
amine oxidase [9,10] reveal the similarity of the overall
fold of these enzymes from various sources and point
to the importance of the channel involved in amine

substrate binding. The domain including the catalytic
region (called D4) exhibits a rather high sequence
similarity. All these features may implicate a similar
Keywords
amine oxidase; substrate docking; substrate
selectivity; substrate-dependent catalytic
mechanism; titratable amino acids
Correspondence
M. Scarpa, Dipartimento di Fisica, Via
Sommarive 14, 38050 Povo-Trento, Italy
Fax: ++39 0461881696
Tel: ++39 0461882029
E-mail:
(Received 13 October 2010, revised 24
December 2010, accepted 2 February 2011)
doi:10.1111/j.1742-4658.2011.08044.x
The structures of copper amine oxidases from various sources show good
similarity, suggesting similar catalytic mechanisms for all members of this
enzyme family. However, the optimal substrates for each member differ,
depending on the source of the enzyme and its location. The structural fac-
tors underlying substrate selectivity still remain to be discovered. With this
in view, we examined the kinetic behaviour of pea seedling amine oxidase
with cadaverine and hexylamine, the first bearing two, and the second only
one, positively charged amino group. The dependence of K
m
and catalytic
constant (k
c
) values on pH, ionic strength and temperature indicates that
binding of the monoamine is driven by hydrophobic interactions. Instead,

binding of the diamine is strongly facilitated by electrostatic factors, con-
trolled by polar side-chains and two titratable residues present in the active
site. The position of the docked substrate is also essential for the participa-
tion of titratable amino acid residues in the following catalytic steps. A
new mechanistic model explaining the substrate-dependent kinetics of the
reaction is discussed.
Abbreviations
AGAO, Arthrobacter globiformis amine oxidase; BSAO, bovine serum amine oxidase; CAD, cadaverine; CuAO, copper amine oxidase; ECAO,
Escherichia coli amine oxidase; HEX, hexylamine; HPAO, Hansenula polimorpha amine oxidase; I, ionic strength;) k
c
, catalytic constant;
PSAO, Pisum sativum amine oxidase; T, temperature; TPQ, 2,4,5-trihydroxyphenylalanine quinone.
1232 FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS
catalytic mechanism for all members of the CuAO
family. The pathway for the reductive half-reaction
has been extensively studied, particularly for the
ECAO [11,12], the HPAO [13] and the BSAO [14,15].
The fundamental reaction steps appear to be similar
for enzymes from various sources [2,15,16]. In particu-
lar, C-H cleavage of the intermediate Schiff base gen-
erated upon amine substrate binding to TPQ appears
to be a crucial step in the mechanism. Dependence of
the kinetic isotope effect on pH [14], site-specific muta-
tions at the active centre [17–19] and the crystal struc-
ture of ECAO in complex with a covalently bound
inhibitor [11], indicate that a fully conserved aspartate
residue (Asp300 for PSAO) serves to abstract the pro-
ton from the Schiff base. This residue also plays a role
in ensuring the correct orientation of the cofactor dur-
ing catalytic turnover [20]. In the course of nucleo-

philic attack, the TPQ ring must be oriented with O5
pointing towards the general base [21], called the ‘pro-
ductive’ conformation. A detailed theoretical study of
the reductive half-reaction of PSAO suggests the possi-
ble role of Lys296, located near TPQ, as a proton
donor [22]. However, the conversion of amine to alde-
hyde groups involves several proton transfer steps and
more than one proton donor or acceptor residue is
involved in catalysis. In spite of their structural simi-
larities, the substrate specificities of CuAOs vary
among enzymes from different sources. In fact, the
best substrates for different CuAOs have different
structure and charge distribution, indicating that sub-
strate-specific interactions govern substrate binding.
The molecular nature of the substrate entry channel
controls substrate binding and subsequent catalysis for
two HPAOs [23]. Electrostatic or hydrophobic forces
have been suggested to drive polyamines (spermine
and spermidine) and long-chain diamines, respectively,
into the BSAO active site [24]. In contrast to the
reductive half-cycle, the oxidative half-cycle is a matter
of debate, and a reaction pathway has been proposed
for plant enzymes, which differs in some steps from
that for mammalian or bacterial enzymes. The oxida-
tive half-cycle of plant enzymes is not rate-limiting
[25,26] and a semiquinone state may be involved in the
catalytic cycle [26,27]. Conversely, in the catalytic cycle
of BSAO and HPAO, the one-electron reduction of di-
oxygen is partially rate-limiting and involves electron
transfer from reduced TPQ to produce superoxide

anion, a reaction intermediate [28].
In this work, we examined the structural factors
underlying substrate specificity and catalytic rates in
copper amine oxidase from pea seedlings. We com-
pared the kinetic behaviour of PSAO with two sub-
strates – the diamine cadaverine (CAD) and the
monoamine hexylamine (HEX) (structures shown in
Fig. 1) – that are very different regarding affinities
(K
m
) and catalytic constants (k
c
). The results from
pH
567891011
k
c
(min
–1
)
0
1000
2000
3000
4000
5000
NH
3
+
NH

3
+
CADAVERINE
NH
3
+
CH
3
HEXYLAMINE
CAD
HEX
pH
567891011
Log (1/K
m
)
1
2
3
4
5
6
CAD
HEX
pH
5678910
Log (kcal·mol
–1
)
3

4
5
6
7
8
9
CAD
HEX
A
B
C
Fig. 1. Effect of pH on kinetic parameters of the CAD (d) and HEX
(
) reductive half-reaction catalyzed by PSAO. (A) k
c
, (B) log(1 ⁄ K
m
)
and (C) log(k
c
⁄ K
m
), versus pH. Curves were obtained by fitting
kinetic parameters to equations for k
c
and K
m
of CAD and HEX, as
reported in the Discussion. The dashed line in panel C is a curve
obtained fitting (k

c
⁄ K
m
)
CAD
according to Dixon’s approach (Eqn 11).
The standard error was within 10% for k
c
and within 15% for K
m
(n = 3).
M. L. Di Paolo et al. Substrate oxidation by amine oxidase
FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS 1233
integration of kinetic studies with docking studies, and
computation of the pK
a
values of the titratable resi-
dues of the active site, suggest that the formation of
the enzyme–substrate complex is precisely regulated by
specific interactions. In particular, the binding of HEX
to the active site is controlled by hydrophobic con-
tacts, whereas the approach of CAD is facilitated by
electrostatic factors, primarily dependent on residues
Glu359 and Glu412. The difference in the binding
mode of the two substrates may modulate the partici-
pation of the residues surrounding TPQ in crucial cat-
alytic steps. In this regard, we confirmed the role of
Asp300 as a general base in the rate-limiting step of
the reaction and indicated Lys296 as playing a sub-
strate-dependent role in the prototropic shift accompa-

nying cleavage of the C
a
-H bond. With the predicted
pK
a
values, a new mechanistic model is proposed
which can satisfactorily explain substrate-dependent
variations in the kinetic data.
Results
A steady-state approach was followed to obtain the
kinetic parameters of PSAO with CAD and HEX as
substrates. In particular, the dependence of kinetic
constants on pH, ionic strength and temperature was
studied to elucidate the electrostatic factors that affect
substrate specificity. Steady-state kinetic experiments
were performed in air-equilibrated solutions at 27 °C.
In these conditions, the rate cannot be affected by the
co-substrate O
2
because the saturation level for O
2
has
been reached. In fact, the concentration of O
2
was
about 0.25 mm [29] and the K
m
(O
2
) values were much

lower: the K
m
(O
2
) was calculated as 17 ± 5 lm when
CAD was used as the saturating amine substate and
the K
m
(O
2
) was calculated to be lower than 2 lm when
HEX was used as the saturating amine, at pH 7.2
(Fig. S1). These low K
m
(O
2
) values match those
obtained with putrescine and benzylamine as sub-
strates, respectively [26]. In addition, according to the
rate constant values for the individual steps reported
by Padiglia et al. [30] for lentil seedling CuAO and to
the kinetic isotope effect reported for PSAO [26], the
oxidative half-cycle is not rate-limiting in air-saturated
solutions and the reductive half-cycle is monitored.
Effect of pH
The dependence of k
c
, log(1 ⁄ K
m
) and log(k

c
⁄ K
m
) val-
ues on pH are shown in Fig. 1. In the pH range
explored, the k
c
values of CAD are always higher than
those of HEX, but become similar at a pH of > 9.5
(Fig 1A). For both substrates, k
c
profiles appear
bell-shaped with the peaks centred at pH values of
$ 7.2 and 9.3, respectively. Regarding the dependence
of 1 ⁄ K
m
on pH, a bell-shaped curve with maximum
values around pH 8.2 was found in the case of CAD,
whereas the HEX K
m
value was independent of pH,
within experimental error (Fig. 1B). The plots of log
(k
c
⁄ K
m
) versus pH are bell-shaped profiles, with the
maximum values centred at about pH 9 and 8 for
CAD and HEX, respectively (Fig. 1C).
Effect of ionic strength

The effect of the ionic environment on the kinetics of
the catalyzed oxidation of CAD and HEX was
measured by varying the ionic strength (I) in the range
20–220 mm, at pH 7.20. Assuming that, for the
enzyme–substrate system under investigation, 1 ⁄ K
m
is
the equilibrium dissociation constant (this point will be
discussed later), the electrostatic effects in PSAO catal-
ysis were studied by varying the ionic strength, and the
data were analysed according to the Debey-Huckel
theory applied to both 1 ⁄ K
m
and k
c
[31]. The plots of
log(k
c
) and log(1 ⁄ K
m
) versus (I)
1 ⁄ 2
were straight lines,
which were fitted to the following equation:
log k ¼ log k
0
þ 2C Â z
A
 z
B

ðIÞ
1=2
; ð1Þ
where k
0
is a kinetic constant or the equilibrium disso-
ciation constant at I = 0, and z
A
and z
B
are the over-
all electrostatic charges of the interacting ionic species
(the substrate and the active site).
Constant C is $ 0.5 in water at 300 K [32]. Values
for the (2*C*z
a
*z
b
) term have been derived for both
substrates and are listed in Table 1. As this term was
found to be close to zero for HEX, the effect of pH
on (2*C*z
a
*z
b
) was tested for CAD only (see Table 1).
The data for HEX suggested that both binding (1 ⁄ K
m
,
see below for a detailed description) and chemical (k

c
)
steps of the catalysis are not controlled by ionic inter-
actions. Conversely, in the case of CAD, a slope
Table 1. Effect of ionic strength on k
c
and 1 ⁄ K
m
at various pH val-
ues. Experimental values of k
c
and 1 ⁄ K
m
versus (I)
1 ⁄ 2
were fitted
to Eqn (1) and values of the linear coefficient (2*C*z
a
*z
b
) are
reported. ND, not determined owing to the low K
m
value.
Substrate
(2*C*z
a
*z
b
)

pH
From log(1 ⁄ K
m
) data From log(k
c
) data
CAD )2.9 ± 0.2 )1.5 ± 0.3 7.2
)1.8 ± 0.4 )1.7 ± 0.1 6.0
ND )1.7 ± 0.1 9.2
HEX 0.2 ± 0.8 )0.1 ± 0.2 7.2
Substrate oxidation by amine oxidase M. L. Di Paolo et al.
1234 FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS
[2*C*z
a
*z
b
= )3] from log(1⁄ K
m
) data was obtained.
The overall CAD charge (z
a
) sensed by the environ-
ment is reported to be z
a
$ 1.3 at pH 7.20 [33] and
this value is expected to be independent of pH up to
about pH 9, where the amino groups can be titrated.
We may thus argue that the positively charged sub-
strate senses an overall charge by about )2 when it
binds into the active site (before the chemical step). At

pH 6, the total negative charge of the active site is
reduced (perhaps one negatively charged group is pro-
tonated) and the slope of the plot of log(1 ⁄ K
m
) versus
(I)
1 ⁄ 2
decreases from about )3 to about )2. The
2*C*z
a
*z
b
of $ )1.6 found for the CAD substrate
from log(k
c
) data in the range of pH explored (pH
6.0–9.2) reflects the fact that the chemical steps of the
reaction are affected by electrostatic interactions
between the negative charges of the enzyme and the
positive charge of the substrate. The amino group in
the substrate tail, which is positively charged in the pH
range explored, may facilitate the correct positioning
of the tail and anchor the substrate at the beginning of
the catalytic cycle.
Effect of temperature
The dependence of k
c
and 1 ⁄ K
m
on temperature, mea-

sured in the range of 290–320 K at pH 7.20, 150 mm
ionic strength, with CAD and HEX as substrates, indi-
cates that these kinetic parameters increase with an
increased temperature, with the exception of the 1 ⁄ K
m
value of CAD, which is independent of the tem-
perature. According to the steady-state approach of
Briggs and Haldane, k
c
is included in K
m
(K
m
=
(k
)1
+ k
c
) ⁄ k
1
): hence, the independence of the K
m
of
HEX from pH and that of the K
m
of CAD from tem-
perature, and the strong dependence of k
c
values of
both substrates on pH and temperature, suggests that

k
c
<< k
)1
, which leads to K
m
@ (k
)1
⁄ k
1
), that is, to
the enzyme–substrate dissociation constant. The deute-
rium kinetic isotope effects investigated by Mukherjee
et al. [26] are consistent with this hypothesis. These
authors observed a strong kinetic isotope effect on
both k
c
and k
c
⁄ K
m
with putrescine and benzylamine as
substrates of PSAO (conversely, if K
m
contains k
c
, the
kinetic isotope effect on k
c ⁄
K

m
should vanish). Accord-
ing to this hypothesis, from the dependence on
temperature (T) of K
m
, we calculated the DH and DS
accompanying the binding of substrate to enzyme
according to the van’t Hoff equation. DH* and DS*,
the enthalpy and entropy of activation accompanying
the formation of the activated complex, were calcu-
lated from the dependence of k
c
on T according to the
transition state theory, and the resulting values are
listed in Table 2. DH* increases with decreasing pH,
whereas -TDS* decreases with decreasing pH, as shown
by the DH* and -TDS* values plotted as a function of
the pH (Fig. 2). Accordingly, the energy cost of the
heterolytic cleavage of the C
a
-H bond is greater at
higher H
+
concentrations, although entropy changes
become less unfavourable. Interestingly, the DH* val-
ues of CAD and HEX show better agreement in the
high-pH range, where the neutral forms of these com-
pounds predominate.
Modelling of substrate–PSAO interactions
Docking CAD into the active site revealed that the

head amino group is located at the bottom of the nar-
row channel and always forms hydrogen bonds with
O5 of TPQ and the carboxylic group of Asp300. There
are three stable conformations for CAD (Fig 3A–C).
Table 2. Thermodynamic parameters of the reductive half-reac-
tion of CAD and HEX by PSAO. Experiments were performed at
pH 7.2 and 150 m
M ionic strength by varying the temperature in
the range 290–320 K. Values of the activation enthalpy (DH*) and
the activation entropy (DS*) were calculated by fitting data of k
c
at
various T to Eqn (12). Values of enthalpy (DH) and entropy (DS)
change were obtained by fitting data of 1 ⁄ K
m
at various tempera-
tures to Eqn (13).
Substrate
Enthalpy (kcalÆmol
)1
) Entropy (calÆmol
)1
ÆK
)1
)
DH*
(from k
c
)
DH

(from 1 ⁄ K
m
)
DS*
(from k
c
)
DS
(from 1 ⁄ K
m
)
CAD 9.1 ± 0.2 0.1 ± 0.1 )25.3 24
HEX 10.9 ± 0.5 2.7 ± 0.4 )20.6 22
pH
56
78
910
Energy (kcal·mol
–1
)
4
6
8
10
12
14
ΔH
#
–TΔS
#

Fig. 2. Effect of pH on heats of activation and activation entropy
for oxidative deamination catalyzed by PSAO. DH* and TDS* values
at various pH values were obtained from ln k
c
versus 1 ⁄ T plots
according to Eqn (12). DH* for CAD (d) and HEX (s), (-TDS*) for
CAD (
) and HEX (h). (-TDS*) values were calculated at 300K.
M. L. Di Paolo et al. Substrate oxidation by amine oxidase
FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS 1235
In the first stable conformation showing the lowest
energy ()13.4 kcalÆmol
)1
) (Fig. 3A), the charged tail
amino group is in contact with polar or negatively
charged residues (Glu412 and Asn386, located at the
bottom of the channel near TPQ). In the second stable
conformation ()13.0 kcalÆmol
)1
), the amino group is
located in a polar pocket composed of Gln108, Ser138
and Ser139 (Fig. 3B). A third stable conformation
()11.8 kcalÆmol
)1
) (Fig. 3C), albeit energetically less
favoured, shows the CAD tail close to Ser138 and
Tyr168, which is hydrogen-bonded to Glu359 of the
other subunit; the hydroxyl group of Tyr168 can form
a hydrogen bond with both Glu359 and the substrate.
In all three conformations, the charged side-chain of

Lys296 is stabilized by forming a salt bridge with
Glu412, and the dihedral angle v
2
of Phe298 is about
)85°, whereas it is about )30° in the original crystal
structure.
The docking simulation of HEX finds two stable
conformations for this substrate with similar binding
energy. In one conformation ()10.8 kcalÆmol
)1
;
Fig. 3D), the head amino group of HEX is located
between TPQ and Asp300, like CAD, and the dihedral
angle v
2
of Phe298 is about )85°. The other conforma-
tion ()10.9 kcalÆmol
)1
; Fig. 3E) shows the amino
group far from Asp300, close to the other side of the
TPQ ring, forming a salt bridge with Glu412 and the
O4 of TPQ, and a hydrogen bond with Asn386. Unlike
the first conformation, the dihedral angle v
2
of Phe298
is about )30°. In both conformations the uncharged
side-chain of Lys296 is hydrogen bonded with the O4
of TPQ.
To determine the charged-state of residues involved
in substrate binding or in catalytic steps, we computed

the pK
a
of the titratable residues in the presence of the
substrate. For the general base candidate Asp300, a
pK
a
value of 8.7 was obtained for the free enzyme,
decreasing to 6.6 with CAD or HEX bound at the
active site. As noted previously for the free enzyme
[32], the large pK
a
shift of this residue in PSAO is
caused by the highly hydrophobic microenvironment
at the enzyme active site. The calculated pK
a
of 6.6
for Asp300 in the active site with substrate bound
match the suggestions for BSAO in an early work by
Fig. 3. Docking of substrates in the PSAO active site. Carbons of the substrate are shown in yellow and carbons of PSAO are shown in
green. Residues that were mobile in docking simulations and residues cited in the text are shown and labelled. Dotted lines: polar contacts.
(A–C) Stable conformations of CAD in the active site; (D, E) stable conformations of HEX. See the text for details.
Substrate oxidation by amine oxidase M. L. Di Paolo et al.
1236 FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS
Klinman et al. [14]. Similar results for the pK
a
value of
the catalytic aspartate with the substrate or inhibitor
in the active site have also been obtained for ECAO
[17,18] and AGAO [19]. In addition, the carboxylic
group of Asp300 forms a hydrogen bond with a TPQ

carbonyl in the crystal structure of PSAO, suggesting
the presence of the protonated form at pH 4.8, the pH
of crystallization. The calculated pK
a
of Lys296
(pK
a
= 8.3) is reduced by more than two pH units
compared with its value in water. The pK
a
values
obtained for Glu359 and Glu412 (7.3 and 5.2, respec-
tively), suggest that these residues change their proton-
ation states in the pH range explored (i.e. by
electrostatic interactions, they may interfere with the
binding of charged substrates). Lastly, a pK
a
of $ 11
was found for Tyr286. It is difficult to assess the error
range of pK
a
values because they have not been experi-
mentally determined in the enzyme. Hence, we estimate
the error range, based on the uncertainty of the
method, as 0.5 pK
a
units [34,35].
pK
a
calculations were also performed with HEX

bound at the active site and the values obtained were
very similar to those with CAD and those reported
above.
Discussion
The above results for PSAO indicate that the binding
of CAD, a substrate which bears one positive charge
on the head and one on the tail, occurs with maximum
efficiency (highest k
c
⁄ K
m
and lowest K
m
values) at a
pH of about 8. According to ionic strength dependence,
binding appears to be driven by the electrostatic inter-
actions occurring between CAD and polar or nega-
tively charged residues located close to the active site.
Based on the modelled structure with CAD bound,
Glu359 and Glu412 favour stable conformations of the
enzyme–substrate complex when negatively charged.
The independence of K
m
of the only head charge-bear-
ing HEX on ionic strength indicates the lack of charge–
charge interactions of this substrate. These observa-
tions, together with the negligible variations of K
m
on
pH, and the positive values of DH and DS, all suggest

that binding of the HEX substrate is primarily driven
by hydrophobic interactions [36]. The high and positive
values of DS of both CAD and HEX (+22 and
+24 cal mol
)1
ÆK
)1
), calculated from the temperature
dependence of the K
m
, suggest that substrate binding is
accompanied by the release of water molecules. Con-
cerning the chemical steps, the bell-shaped profile of
the k
c
of CAD and HEX versus pH (Fig. 1B) indicates
the involvement of at least two acid–base couples, (B
1
-
H
+
⁄ B
1
) and (B
2
-H
+
⁄ B
2
), in the rate-determining step,

like the two-protonation state model of Tipton and
Dixon [37]. Fig. 1B shows that the pK
a
values of B
1
and B
2
are substrate dependent; alternatively, and more
probably, different residues behave as B
1
and B
2
,
depending on the structure of the interacting substrate.
According to the above results and the fundamental
steps of the reaction described in the literature, shown
in Fig. 4A, we propose a kinetic model (for details see
Doc. S1), in which the only charged forms of CAD are
considered as reactive species, because the charged
amine groups favour interactions with the active site.
Hence, we included [S]
R
= [SH
+
] + [SH
2
2+
] for CAD.
Conversely, ([S]
R

= [S] + [SH
+
]) was considered for
HEX because hydrophobic interactions with the active
site prevail. However, we assumed that, in both sub-
strates, the attacking amino group was neutral at the
beginning of the catalytic cycle so that the nucleophilic
attack on TPQ could take place [2]. During the sub-
strate entry (not explicitly shown in the simplified
scheme of Fig. 4A) the CAD tail is addressed towards
stable enzyme–complex conformations by two nega-
tively charged residues (Glu359 and Glu412) and by
polar residues (Asn386, Ser138 and Tyr168), as shown
in Fig. 3. The two charged residues are titrated in the
pH range explored and facilitate interaction between
enzyme and substrate. The kinetic rate constant, k
1
,of
the recognition step, which leads to the formation of
the enzyme–substrate complex (before the chemical
events), may be written as:
k
1
¼
~
k
1
 e
À
d

1
RT
K
D1
K
D1
þ½H
þ

ðÞ
À
d
2
RT
K
D2
K
D2
þ½H
þ

ðÞ
no
; ð2Þ
where the energy of the electrostatic interaction (d
1
and d
2
) of the substrate with two titratable residues,
D1 and D2 (probably Glu359 and Glu412), with ioni-

zation constants of K
D1
and K
D2
, is explicitly reported.
The terms K
D1
⁄ (K
D1
+[H
+
]) and K
D2
⁄ (K
D2
+[H
+
])
are weighting factors taking into account the molar
fraction of D1 and D2 in the deprotonated state. In
the case of HEX the ionization contributions to k
1
vanish.
~
k
1
includes all the other energy terms contrib-
uting to the kinetic constant (i.e. the contribution of
the electrostatic interaction between substrate and
polar residues and of the hydrophobic interaction).

The fundamental points of our approach describing
the catalytic events are as follows.
1. The role played by proton-exchanging residues on
the recognition step and on the chemical reaction is
explicitly introduced both in K
m
by Eqn (2), showing
residues D1 and D2, and in k
c
by assuming the pres-
ence of B1 and B2 residues.
2. Small differences in substrate structure produce a
different enzyme complex, so that the enzyme residues
M. L. Di Paolo et al. Substrate oxidation by amine oxidase
FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS 1237
involved in catalysis, in both recognition and reaction
steps, are substrate dependent (see also Fig. 4A show-
ing the fundamental steps of the reaction pathway).
The position of the TPQ intermediate (the ketimine
I
±
) is consequently modified.
This hypothesis was confirmed by docking computa-
tions, which indicated that deprotonated Lys296 points
towards TPQ only in the stable conformations of the
HEX–enzyme complex. Conversely, the charge–charge
interaction facilitating the binding and positioning of
A
B
Fig. 4. Proposed mechanism of the reduc-

tive half-reaction catalyzed by PSAO with
CAD and HEX as substrates. (A) Fundamen-
tal steps of the reductive half-reaction. B
1
and B
2
, two titratable groups participating in
the rate-limiting step; E, enzyme; I
±
, Schiff
base ketimine form; I
þ
À
, Schiff base aldimine
form; P, product; S, substrate (CAD or
HEX). (B) Concerted prototropic shift occur-
ring during the rate-limiting step. Involve-
ment of different proton donors and
acceptors if CAD or HEX is the substrate.
Substrate oxidation by amine oxidase M. L. Di Paolo et al.
1238 FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS
CAD also helps to accelerate the chemical events lead-
ing to an increase in k
c
. The importance of this inter-
action is also supported by the similar behaviour of
CAD and HEX above pH 9.5, when both substrates
are present in their neutral form.
3. The heterolytic cleavage of the C
a

-H bond of the
amine is assumed to control the k
c
of the reductive
half-step, as supported in the literature [2,23]. The con-
certed prototropic shift converting the Schiff base from
the ketimine form (I
±
) to the aldimine form (I
þ
À
)is
assisted by two acid–base couples: (B
1
-H
+
⁄ B
1
) and
(B
2
-H
+
⁄ B
2
), which interact simultaneously with the
Schiff base (see Fig. 4B). From our data it appears
that the identity of these residues is substrate depen-
dent, which may account for the differences in the pH
dependence of the k

c
values (Fig. 1A).
On the basis of the three points described above,
and assuming that the deprotonation of the head
amino group is not rate-limiting, the following equa-
tions were derived for CAD (the detailed kinetic model
is reported in Doc. S1):
v ¼ k
2
E½
0
S½
0
S½
0
þ
k
À1
þk
2
k
1
Â
K
2
S
þK
S
H
þ

½þH
þ
½
2
K
S
H
þ
½þH
þ
½
2
no
Â
1 þ a
H
þ
½
K
B1
þ b
K
B2
H
þ
½

1 þ
H
þ

½
K
B1
þ
K
B2
H
þ
½
ð3Þ
and for HEX, respectively:
v ¼
k
0
2
S½
0
E½
0
S½
0
þ
k
0
À1
þk
0
2
k
0

1

Â
1
1 þ
H
þ
½
K
B1
þ
K
B2
H
þ
½
; ð4Þ
where [S ]
0
and [E]
0
are the total concentrations of the
substrate and enzyme, respectively.
K
B1
and K
B2
are the ionization constants of the two
general bases which control k
c

, and a and b are empir-
ical constants representing the partial activity at extre-
mal pH [37]; k
1
is given by Eqn (2).
According to Eqns (3,4), the experimental data of
Fig. 1 were fitted to the following equations (solid lines
of Fig. 1):
k
cðCADÞ
¼
k
2
1 þ a
H
þ
½
K
B1
þ b
K
B2
H
þ
½

1 þ
H
þ
½

K
B1
þ
K
B2
H
þ
½
ð5Þ
logð1=K
mðCADÞ
Þ¼ Àlog
k
À1
þ k
2
~
k
1
 e
À
d
1
RT
K
D1
ðK
D1
þ H
þ

½
Þ
À
d
2
RT
K
D2
ðK
D2
þ H
þ
½
Þ
no
2
6
4
Â
K
2
S
þ K
S
H
þ
½þH
þ
½
2

K
S
H
þ
½þH
þ
½
2
()#
ð6Þ
logðk
c
=K
mðCADÞ
Þ¼ Àlog
k
À1
þ k
2
~
k
1

þ log e
À
d
1
RT
K
D1

ðK
D1
þ H
þ
½
Þ
À
d
2
RT
K
D2
ðK
D2
þ H
þ
½
Þ
no
"#
À log
K
2
S
þK
S
H
þ
½þH
þ

½
2
K
S
H
þ
½þH
þ
½
2
1 þ
H
þ
½
K
B1
þ
K
B2
H
þ
½
2
6
4
3
7
5
ð7Þ
k

cðHEXÞ
¼
k
0
2
1 þ
½H
þ

K
B1
þ
K
B2
½H
þ

ð8Þ
logðk
c
=K
mðHEXÞ
Þ¼log
k
0
2
1 þ
H
þ
½

K
B1
þ
K
B2
H
þ
½
0
@
1
A
: ð9Þ
The resulting d
1
, d
2
and pK
a
values are listed in
Table 3.
Equation (8) is equivalent to the Tipton and Dixon
equation for k
c
(according to their ‘Simplified reaction
scheme’ [38]), where the a and b factors [37] may be
included to obtain Eqn (5).
The Dixon’s models [39], which are usually utilized
to predict pK
a

values from kinetic data, were used for
comparison. In the case of CAD, the fit of K
m
and
k
c
⁄ K
m
were performed with a three pK
a
model, fitting
a bell-shaped curve with an increase with two pK
a
val-
ues and a decrease with one pK
a
value.
log 1=K
m
ðÞ¼log 1=K
m
0
ðÞÀlog H
þ
½
2
=ðK
1
Á K
2

Þ

þ H
þ
½=K
2
þ 1 þ K
3
= H
þ
½Þ ð10Þ
log k
c
=K
m
ðÞ¼log k
c
=K
m
ðÞ
0
À log H
þ
½
2
=ðK
1
Á K
2
Þ


þ H
þ
½=K
2
þ 1 þ K
3
= H
þ
½Þ ð11Þ
The estimated pK
a
values according to Eqns (10, 11)
are reported in the last column of Table 3.
A good match was found between the two sets of
data, that is pK
a
values according to Dixon and to the
model we are proposing. However, the models of
Dixon do not estimate the contributions to the Gibbs
energy of the recognition step due to D
1
and D
2
(d
1
and d
2
).
The equation for (k

c
⁄ K
m
)
HEX
(a two-pK
a
model),
according to the approach of Dixon, is formally equiv-
alent to Eqn (9).
In addition, from Table 3 it appears that the pK
a
values obtained by the experimental data are in good
agreement also with the computed pK
a
values reported
in the Modelling of substrate-PSAO interactions sec-
tion. In particular, D
1
could be Glu412 (computed
M. L. Di Paolo et al. Substrate oxidation by amine oxidase
FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS 1239
pK
a
= 5.2) and D
2
could be identified with Glu359
(computed pK
a
= 7.2).

The pK
a
values calculated from k
c
with the CAD
substrate are also in accordance with those obtained
by Pec et al. [40] for the similar, but more rigid, 1,4-
diamino-2-butene substrate (pK
a
values of 6.9 and 8.1
were obtained from the fit of the k
c
data).
The structure of the catalytic site and the calculated
and experimentally obtained pK
a
values identify
Asp300 (pK
a
= 6.6) and Lys296 (pK
a
= 8.3) as cata-
lytically important residues, with pK
a
values falling
into the pH range delimiting the k
c
bells. Based on the
k
c

versus pH profiles and on the docking studies,
which show a Lys296 orientation that is substrate
dependent (Lys296 forms a salt bridge with TPQ and
with Glu412 when HEX or CAD, respectively, are in
the active site), we proposed the role for Lys296 as a
proton donor in the case of CAD and as a proton
acceptor in the case of HEX. Asp300 is the proton
acceptor candidate in the case of CAD. The position
of Tyr286 indicates this residue as a possible candidate
for donating a proton (Fig. 4B). Its role in proton
transfer has already been suggested by Hevel et al. [13]
on HPAO.
The results from Pietrangeli et al. [41] with two ali-
phatic amines (putrescine and spermidine) and four aro-
matic amines have been interpreted in terms of
hydrophobic interactions prevailing over polar interac-
tions in PSAO. Our results partially match those of
these authors, in that the substrate contains a hydro-
phobic tail. However the tail amino group of CAD not
only affects K
m
but increases, in orders of magnitude, k
c
at the optimum pH value. Consequently, the electro-
static-driven docking of CAD appears to be crucial for
the substrate preference of PSAO. Conversely, if the
electrostatic contribution is lacking, increased flexibility
of the substrate Schiff base would be expected. A similar
effect (although of hydrophobic rather than of electro-
static nature) was reported by Taki et al. [42] studying

the stereo-selectivity of a bacterial amine oxidase.
In conclusion, in a combination of kinetic, structural
and computational procedures, this study shows that
the substrate-specific interactions underlying the selec-
tivity of PSAO not only affect the binding mode of the
amine in the active site, but also the identity of the res-
idues recruited in the catalytic steps. In particular, the
new role of Lys206 is proposed in the catalytic cycle.
Because this Lys is a conserved residue in plant
CuAOs and has been proposed to play a role in the
formation of TPQ
sq
upon oxidative deamination of its
side-chain [43], future study of site-directed mutagene-
sis will be necessary to confirm our findings and to
have a better understanding of the structural factors
controlling substrate preferences and catalysis of
CuAOs, enzymes with many still unknown physio-
logical functions.
Experimental procedures
Enzyme purification and activity testing
All reagents were from Fluka (Milan, Italy). PSAO was
purified from Pisum sativum seedlings according to Vianello
et al. [44], reaching a final specific activity of 1.6 lkatÆmg
)1
.
Initial-rate measurements were carried out by monitoring
H
2
O

2
production using a peroxidise–cytochrome c-coupled
assay [45]. Kinetic runs were performed at 27 °C, in various
experimental conditions, particularly at variable amine sub-
strate concentrations, pH values (range 5.20–10.20) and
ionic strength (20–220 mm), equilibrated with air. Steady-
state kinetic parameters (k
c
and K
m
) were calculated from
Table 3. Ionization constants and energy contributions from the
pH profile of PSAO kinetic parameters. Column 3: pK
a
values and
free-energy contributions (d
1
and d
2
) were obtained by fitting exper-
imental data of k
c
and K
m
or pseudo-first-order k
c
⁄ K
m
constants as
a function of pH according to the equations described in the Dis-

cussion. HEX: k
c
(Eqn 8) and k
c
⁄ K
m
(Eqn 9) fitting were obtained
leaving all unknown parameters (i.e. K
B1
and K
B2
) floating. CAD: k
c
(Eqn 5) fitting was obtained leaving all unknown parameters float-
ing; in the fitting of K
m
(Eqn 6) and k
c
⁄ K
m
(Eqn 7), K
D1
,K
D2
, d
1
and
d
2
terms were left to float but pK

B1
= 6.66 and pK
B2
= 8.30 were
maintained fixed, as calculated from the k
c
data; pK
s
= 10 was also
maintained fixed. Column 4: pK
a
values from the dependence on
pH of K
m
(Eqn 10) and k
c
⁄ K
m
(Eqn 11) using CAD as a substrate,
according to Dixon’s model [39].
Substrate Kinetic
parameter
pK
a
according to the
proposed model
pKa according to
Dixon’s model
CAD k
c

pK
B1
= 6.66 ± 0.15
pK
B2
= 8.30 ± 0.11
CAD K
m
pK
D1
= 5.37 ± 0.32 pK
D1
= 5.59± 0.31
pK
D2
= 6.90 ± 0.29 pK
D2
= 7.23 ± 0.28
d
1
= )3.97
± 0.79 kcalÆmol
)1
d
2
= )1.21
± 0.46 kcalÆmol
)1
pK
S

= 9.95 ± 0.12 pK
S
= 10.04 ± 0.13
k
c
⁄ K
m
pK
D1
= 5.22 ± 0.62 pK
D2
= 6.16 ± 0.61
pK
D2
= 7.28 ± 0.26 pK
D2
= 7.14 ± 0.28
d
1
= )2.5
± 1.50 kcalÆmol
)1
d
2
= )1.20
± 0.28 kcalÆmol
)1
pK
S
= 10.0 (fixed) pK

S
= 8.95 ± 0.34
HEX k
c
pK
B1
= 8.41 ± 0.17
pK
B2
= 10.36 ± 0.23
k
c
⁄ K
m
pK
B1
= 8.30 ± 0.18
pK
B2
= 10.00 ± 0.35
Substrate oxidation by amine oxidase M. L. Di Paolo et al.
1240 FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS
nonlinear fitting of the reaction rate plots to the Michaelis–
Menten equation using sigmaplot 2004, Version 9.01 (Sy-
stat Software Inc., Richmond, CA, USA). Michaelis–Men-
ten behaviour was observed independently of substrate, pH
and ionic strength.
Experiments were performed in solutions containing
25 mm buffer and 125 mm NaCl at various pH values. The
buffers used were: sodium acetate (pH 5.2–5.6), Mes (pH

5.6–6.4), Mops (pH 6.61–7.03), Hepes (pH 8.00–8.65),
sodium borate (pH 8.71–9.71) and sodium carbonate (pH
9.71–10.20). Kinetic measurements performed in these buf-
fers at overlapping pH values gave identical results within
the experimental error, excluding specific salt effects.
Experiments were performed at pH 7.20, in solution con-
taining 25 mm Hepes at various ionic strengths (10–200 mm
NaCl was added).
The heat of activation (DH*) and entropy (DS*) were
obtained by measuring the effect of temperature on k
c
,
according to the law:
ln k
c
¼ ln j
k
B
T
h
þ
DSÃ
R
À
DHÃ
RT
;
where DH* is the heat of activation, DS* is the entropy of
activation, k
B

is the Boltzmann constant, h is the Plank
constant, R is the gas constant and j is the transmission
coefficient. As j is usually close to unity [46] this equation
simplifies into:
ln k
c
¼ ln
k
B
T
h
þ
DSÃ
R
À
DHÃ
RT
: ð12Þ
The changes in enthalpy (DH) and entropy (DS) of the
binding process were obtained by measuring the effect of
temperature on K
m
, according to the equation:
ln
1
K
m
¼
DS
R

À
DH
RT
; ð13Þ
assuming that 1 ⁄ K
m
values are the association constants of
the enzyme–substrate complex and D H and DS are the ther-
modynamic parameters of enzyme–substrate complex for-
mation.
The constants k
c
and K
m
at various temperatures were
calculated from Michaelis–Menten plots obtained in the
range 290–320 K.
Computational details
We studied the binding modes of CAD and HEX by means
of docking simulation in the PSAO active site. The crystal
structure of free PSAO with the Protein Data Bank code
1KSI [4] was used as a starting model for all calculations.
In this structure the TPQ ring adopts a nonproductive con-
formation (i.e. O2 of TPQ points towards Asp300 and O5
points towards the copper ion cofactor) [22]. Hence, to gen-
erate an appropriate model for the reaction, the TPQ ring
was rotated by 180°.
As the two subunits in PSAO operate simultaneously,
but not cooperatively [47], substrate docking was simulated
only in subunit A. AutoDockTools version 1.5.2 (the

Scripps Research Institute, La Jolla, CA, USA) was used to
add polar hydrogens to the PSAO crystal structure and to
assign Gasteiger charges to the atoms, with the exception
of TPQ, the charges of which were calculated using the
petra web server [ />software/petra]. autodock 4 software was used to perform
docking simulations, employing the Lamarckian genetic
algorithm [48]. Default settings were used for docking
parameters. Other details are available in Doc. S2.
As previously described [33], the active site of PSAO is
extremely hydrophobic, and therefore in order to account
properly for the pK
a
shift of titratable residues at the cata-
lytic centre, a microenvironment-dependent method had to
be applied. Hence, the pK
a
values of titratable active-site
residues in the presence of various substrates were calcu-
lated using the screened Coulomb potential method, with
microenvironment-dependent dielectric screening functions
[34,35]. (Other details can be found in Doc. S2.)
Acknowledgements
This work was partly funded by Istituto Nazionale Bio-
strutture Biosistemi (Rome, Italy) and by Hungarian
Research Fund (OTKA) K72579, M.F for Bolyai
Ja
´
nos fellowship.
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Supporting information
The following supplementary material is available:
Doc. S1. Kinetic model formulation.
Doc. S2. Computational details.
Fig. S1. Reaction rates versus oxygen concentration.
This supplementary material can be found in the
online version of this article.
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M. L. Di Paolo et al. Substrate oxidation by amine oxidase
FEBS Journal 278 (2011) 1232–1243 ª 2011 The Authors Journal compilation ª 2011 FEBS 1243