All these features may implicate a similar Keywords amine oxidase; substrate docking; substrate selectivity; substrate-dependent catalytic mechanism; titratable amino acids Correspondenc
Trang 1oxidation catalysed by pea seedling amine oxidase
Maria Luisa Di Paolo1, Michele Lunelli2, Monika Fuxreiter2,3, Adelio Rigo1, Istvan Simon3
and Marina Scarpa2
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:
RCH2NHþ3þ O2þ H2O! RCHO þ NHþ4 þ H2O2
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: marina.scarpa@unitn.it
(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 Km and catalytic constant (kc) 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.
Trang 2catalytic 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 (Km) and catalytic constants (kc) The results from
pH
kc
–1 )
0 1000 2000 3000 4000 5000
NH 3 N
H3 + CADAVERINE
NH 3 C
H 3 HEXYLAMINE
CAD
HEX
pH
Km
1 2 3 4 5 6
CAD
HEX
pH
–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) kc, (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).
Trang 3integration of kinetic studies with docking studies, and
computation of the pKa 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 Ca-H bond With the predicted
pKa 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 27C
In these conditions, the rate cannot be affected by the
co-substrate O2because the saturation level for O2has
been reached In fact, the concentration of O2 was
about 0.25 mm [29] and the Km(O2) values were much
lower: the Km(O2) was calculated as 17 ± 5 lm when
CAD was used as the saturating amine substate and
the Km(O2) was calculated to be lower than 2 lm when
HEX was used as the saturating amine, at pH 7.2
(Fig S1) These low Km(O2) 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 kc, log(1⁄ Km) and log(kc⁄ Km)
val-ues on pH are shown in Fig 1 In the pH range
explored, the kcvalues of CAD are always higher than
those of HEX, but become similar at a pH of > 9.5
(Fig 1A) For both substrates, kc profiles appear
bell-shaped with the peaks centred at pH values of
7.2 and 9.3, respectively Regarding the dependence
of 1⁄ Km on pH, a bell-shaped curve with maximum values around pH 8.2 was found in the case of CAD, whereas the HEX Km value was independent of pH, within experimental error (Fig 1B) The plots of log (kc⁄ Km) 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⁄ Km 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⁄ Kmand kc [31] The plots of log(kc) and log(1⁄ Km) versus (I)1⁄ 2 were straight lines, which were fitted to the following equation:
log k¼ log k0þ 2C zA zBðIÞ1=2; ð1Þ where k0 is a kinetic constant or the equilibrium disso-ciation constant at I = 0, and zA and zB 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*za*zb) 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*za*zb) was tested for CAD only (see Table 1) The data for HEX suggested that both binding (1⁄ Km, see below for a detailed description) and chemical (kc) 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 kcand 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(kc) data
Trang 4[2*C*za*zb=)3] from log(1 ⁄ Km) data was obtained.
The overall CAD charge (za) sensed by the
environ-ment is reported to be za 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⁄ Km) versus
(I)1⁄ 2 decreases from about )3 to about )2 The
2*C*za*zb of )1.6 found for the CAD substrate
from log(kc) 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 kcand 1⁄ Km 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⁄ Km
value of CAD, which is independent of the
tem-perature According to the steady-state approach of
Briggs and Haldane, kc is included in Km (Km=
(k)1+ kc)⁄ k1): hence, the independence of the Km of
HEX from pH and that of the Kmof CAD from
tem-perature, and the strong dependence of kc values of
both substrates on pH and temperature, suggests that
kc<< k)1, which leads to Km@ (k)1⁄ k1), 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 kcand kc⁄ Kmwith putrescine and benzylamine as
substrates of PSAO (conversely, if Km contains kc, the
kinetic isotope effect on kc⁄Kmshould vanish)
Accord-ing to this hypothesis, from the dependence on
temperature (T) of Km, 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 kcon 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 Ca-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)1ÆK)1) DH*
(from k c )
DH (from 1⁄ K m )
DS*
(from k c )
DS (from 1 ⁄ K m )
pH
–1 )
4 6 8 10 12 14
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 kc 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.
Trang 5In 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 v2 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 v2of 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 v2 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 pKa of the titratable residues in the presence of the substrate For the general base candidate Asp300, a
pKa 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 pKa shift of this residue in PSAO is caused by the highly hydrophobic microenvironment
at the enzyme active site The calculated pKa 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.
Trang 6Klinman et al [14] Similar results for the pKavalue 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 pKa of Lys296
(pKa= 8.3) is reduced by more than two pH units
compared with its value in water The pKa 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 pKa of 11
was found for Tyr286 It is difficult to assess the error
range of pKavalues 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 pKaunits [34,35]
pKa 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 kc⁄ Km and lowest Km 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 Kmof 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 Km 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)1ÆK)1), calculated from the temperature
dependence of the Km, suggest that substrate binding is
accompanied by the release of water molecules
Con-cerning the chemical steps, the bell-shaped profile of
the kcof CAD and HEX versus pH (Fig 1B) indicates
the involvement of at least two acid–base couples, (B1
-H+⁄ B1) and (B2-H+⁄ B2), in the rate-determining step,
like the two-protonation state model of Tipton and Dixon [37] Fig 1B shows that the pKa values of B1 and B2are substrate dependent; alternatively, and more probably, different residues behave as B1 and B2, 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+] + [SH22+] 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, k1, of the recognition step, which leads to the formation of the enzyme–substrate complex (before the chemical events), may be written as:
k1¼ ~k1 e
d1 RT KD1 KD1þ½Hþ
ð ÞRTd2
KD2 KD2þ½Hþ
where the energy of the electrostatic interaction (d1 and d2) of the substrate with two titratable residues, D1 and D2 (probably Glu359 and Glu412), with ioni-zation constants of KD1and KD2, is explicitly reported The terms KD1⁄ (KD1+ [H+]) and KD2⁄ (KD2+ [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 k1 vanish ~k1includes 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 Km by Eqn (2), showing residues D1 and D2, and in kc 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
Trang 7involved 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 B1 and B 2 , two titratable groups participating in the rate-limiting step; E, enzyme; I ± , Schiff base ketimine form; I þ , Schiff base aldimine form; P, product; S, substrate (CAD or HEX) (B) Concerted prototropic shift occur-ring duoccur-ring the rate-limiting step Involve-ment of different proton donors and acceptors if CAD or HEX is the substrate.
Trang 8CAD also helps to accelerate the chemical events
lead-ing to an increase in kc 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 Ca-H bond of the
amine is assumed to control the kc 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 (Iþ) is
assisted by two acid–base couples: (B1-H+⁄ B1) and
(B2-H+⁄ B2), 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 kcvalues (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):
v¼ k2
E
½ 0½ S0 S
½ 0þk1 þk 2
k1 K2þKS ½ H þ þ H ½ þ 2
K S ½ H þ þ H ½ þ 2
þ a½HKþ
B1 þ bKB2
H þ
½
1þ½KHþ
B1þK B2
H þ
½ ð3Þ
and for HEX, respectively:
v¼ k
0
2½ S0½ E0 S
½ 0þ k01 þk 0
2
k 0 1
1þ½KHþ
B1 þK B2
H þ
½
where [S]0 and [E]0 are the total concentrations of the
substrate and enzyme, respectively
KB1 and KB2 are the ionization constants of the two
general bases which control kc, and a and b are
empir-ical constants representing the partial activity at
extre-mal pH [37]; k1is 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):
kcðCADÞ¼k2 1þ a
H þ
½
KB1 þ bK B2
H þ
½
1þ½KHþ
B1 þK B2
H þ
½
ð5Þ
logð1=KmðCADÞÞ ¼ log k1þ k2
~
k1 e
d1 RT
KD1 ðKD1þ Hþ ½ Þ RTd2 KD2
ðKD2þ Hþ ½ Þ
2 6
2þ KS½Hþ þ H½ þ2
KS½Hþ þ H½ þ2
ð6Þ
logðkc=KmðCADÞÞ ¼ log k1~þ k2
k1
þ log e
d1 RT
KD1 ðKD1þ Hþ ½ Þ RTd2 KD2
ðKD2þ Hþ ½ Þ
log
K 2 þK S ½ H þ þ H ½ þ 2
K S ½ H þ þ H ½ þ 2
1þ½HKþ
B1 þ K B2
H þ
½
2 6
3
0 2
1þ½HKþ
B1 þ K B2
½H þ
ð8Þ
logðkc=KmðHEXÞÞ ¼ log k
0 2
1þ½HKþ
B1 þ KB2
H þ
½
0
@
1
The resulting d1, d2 and pKa values are listed in Table 3
Equation (8) is equivalent to the Tipton and Dixon equation for kc(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 pKa values from kinetic data, were used for comparison In the case of CAD, the fit of Km and
kc⁄ Km were performed with a three pKa model, fitting
a bell-shaped curve with an increase with two pKa val-ues and a decrease with one pKavalue
log 1=Kð mÞ ¼ log 1=Kð m 0Þ log H½ þ2=ðK1 K2Þ
þ H½ þ=K2þ 1 þ K3=½HþÞ ð10Þ
log kð c=KmÞ ¼ log kð c=KmÞ0 log H½ þ2=ðK1 K2Þ
þ H½ þ=K2þ 1 þ K3=½HþÞ ð11Þ
The estimated pKa 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 pKavalues 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 D1 and D2 (d1 and d2)
The equation for (kc⁄ Km)HEX (a two-pKa model), according to the approach of Dixon, is formally equiv-alent to Eqn (9)
In addition, from Table 3 it appears that the pKa values obtained by the experimental data are in good agreement also with the computed pKa values reported
in the Modelling of substrate-PSAO interactions sec-tion In particular, D1 could be Glu412 (computed
Trang 9pKa= 5.2) and D2 could be identified with Glu359
(computed pKa= 7.2)
The pKa values calculated from kc 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 (pKa values of 6.9 and 8.1
were obtained from the fit of the kcdata)
The structure of the catalytic site and the calculated
and experimentally obtained pKa values identify
Asp300 (pKa = 6.6) and Lys296 (pKa= 8.3) as
cata-lytically important residues, with pKa values falling
into the pH range delimiting the kcbells Based on the
kc 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 Kmbut increases, in orders of magnitude, kc
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 TPQsq 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
Initial-rate measurements were carried out by monitoring
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
Table 3 Ionization constants and energy contributions from the
pH profile of PSAO kinetic parameters Column 3: pK a values and
free-energy contributions (d1and d2) were obtained by fitting
exper-imental data of kcand Kmor pseudo-first-order kc⁄ K m constants as
a function of pH according to the equations described in the
Dis-cussion HEX: kc (Eqn 8) and kc⁄ K m (Eqn 9) fitting were obtained
leaving all unknown parameters (i.e KB1and KB2) floating CAD: kc
(Eqn 5) fitting was obtained leaving all unknown parameters
float-ing; in the fitting of Km(Eqn 6) and kc⁄ K m (Eqn 7), KD1, KD2, d1and
d2terms were left to float but pKB1= 6.66 and pKB2= 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 Km(Eqn 10) and kc⁄ K m (Eqn 11) using CAD as a substrate,
according to Dixon’s model [39].
Substrate Kinetic
parameter
pKaaccording to the proposed model
pKa according to Dixon’s model
pK B2 = 8.30 ± 0.11 CAD Km pKD1= 5.37 ± 0.32 pKD1= 5.59± 0.31
pKD2= 6.90 ± 0.29 pKD2= 7.23 ± 0.28
d 1 = )3.97
± 0.79 kcalÆmol)1
d2= )1.21
± 0.46 kcalÆmol)1
pK S = 9.95 ± 0.12 pK S = 10.04 ± 0.13
kc⁄ K m pKD1= 5.22 ± 0.62 pKD2= 6.16 ± 0.61
pKD2= 7.28 ± 0.26 pKD2= 7.14 ± 0.28
d 1 = )2.5
± 1.50 kcalÆmol)1
d2= )1.20
± 0.28 kcalÆmol)1
pKS= 10.0 (fixed) pKS= 8.95 ± 0.34
pK B2 = 10.36 ± 0.23
k c ⁄ K m pK B1 = 8.30 ± 0.18
pKB2= 10.00 ± 0.35
Trang 10nonlinear 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
according to the law:
where DH* is the heat of activation, DS* is the entropy of
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 kc¼ lnkBT
h þ
DS
DH
The changes in enthalpy (DH) and entropy (DS) of the
binding process were obtained by measuring the effect of
the enzyme–substrate complex and DH and DS are the
ther-modynamic parameters of enzyme–substrate complex
for-mation
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
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
cata-lytic centre, a microenvironment-dependent method had to
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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