APase displays higher activity in the presence of Mg2+, as binding of Mg2+increases the rate of conformational change.. Therefore, Mg2+ activation was studied using an enzyme fully satur
Trang 1Dimer asymmetry and the catalytic cycle of alkaline phosphatase
Stjepan Orhanovic´ and Maja Pavela-Vrancˇicˇ
Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, Croatia
Although alkaline phosphatase (APase) from Escherichia
colicrystallizes as a symmetric dimer, it displays deviations
from Michaelis–Menten kinetics, supported by a model
describing a dimeric enzyme with unequal subunits
[Orha-novic´ S., Pavela-Vrancˇicˇ M and Flogel-Mrsˇic´ M (1994)
Acta Pharm 44, 87–95] The possibility, that the observed
asymmetry could be attributed to negative cooperativity in
Mg2+ binding, has been examined The influence of the
metal ion content on the catalytic properties of APase from
E colihas been examined by kinetic analyses An activation
study has indicated that Mg2+enhances APase activity by a
mechanism that involves interactions between subunits The
observed deviations from Michaelis–Menten kinetics are
independent of saturation with Zn2+or Mg2+ions,
sug-gesting that asymmetry is an intrinsic property of the dimeric
enzyme In accordance with the experimental data, a model
describing the mechanism of substrate hydrolysis by APase has been proposed The release of the product is enhanced by
a conformational change generating a subunit with lower affinity for both the substrate and the product In the course
of the catalytic cycle the conformation of the subunits alternates between two states in order to enable substrate binding and product release APase displays higher activity
in the presence of Mg2+, as binding of Mg2+increases the rate of conformational change A conformationally con-trolled and Mg2+-assisted dissociation of the reaction product (Pi) could serve as a kinetic switch preventing loss of
Piinto the environment
Keywords: metalloenzymes; conformational change; sub-unit interactions; enzyme asymmetry; phosphate meta-bolism
Most unresolved questions, relating to the catalytic
mech-anism of alkaline phosphatase (APase, E.C 3.1.3.1),
con-cern the influence of conformational changes and allosteric
interactions on catalytic efficiency Crystallographic
ana-lysis has shown that APase from E coli has three metal
binding sites [1] Both zinc ions in the active site are
essential for activity [2], whereas magnesium alone does not
activate the apoenzyme but increases the activity of the
Zn2+-containing APase [3,4] Significant cooperative
inter-actions have been detected during metal-ion binding,
positive for the binding of Zn2+ to the M1 site, and
negative for the binding of the activating cations to the M3
site [5,6] Phosphomonoester hydrolysis and
transphos-phorylation, catalyzed by APase, proceeds through a
covalent serine-phosphate intermediate [7,8] Dissociation
of the reaction product, Pi, is rate limiting at alkaline pH
In the case of Pihydrolysis, phosphorylation of Ser102 is slow enough to become the rate-determining step [9] APase activity increases in the presence of phosphate-accepting alcohols The rate of Piformation is unchanged, indicating that the newly generated phosphomonoester dissociates much faster than Pi It has been suggested that
Pi is bound to the active site in form of a dianion [9], however, the slow dissociation of Pi, and the slow phosphorylation of Ser102 by Pi, are both in accordance with Pibinding in form of a trianion
The crystal structure of APase from E coli has shown that metal–metal distances vary slightly between neighbor-ing subunits, but the significance of these differences is not clear The Mg2+binding site is not close enough to allow for the direct participation of Mg2+in phosphomonoester hydrolysis [9] The crystal structure of APase in complex with Pi(APasePi), determined by Stec et al differs from that resolved by Kim (1990), particularly with respect to the Ser102 conformation and the nature of the metal ion bound
to the M3 site [10] The APasePi structure displays an increased mobility of the active site with pronounced anisotropy for the metal ions and the Arg166 side-chain [10]
APase belongs to a large group of enzymes displaying deviations from Michaelis–Menten kinetics, resembling negative cooperativity and half-of-the-sites reactivity [11–15] Although half-of-the-sites reactivity is a widespread phenomenon among oligomeric enzymes, a satisfactory explanation describing the advantage of such kinetic properties is still lacking [16,17] Steady state kinetics, resulting in curved Lineweaver–Burk plots, did not agree
Correspondence to M Pavela-Vrancˇicˇ, Department of Chemistry,
Faculty of Natural Sciences, Mathematics and Education,
University of Split, N Tesle 12, 21000 Split, Croatia.
Fax: + 385 21 385431, Tel.: + 385 21 385009,
E-mail: pavela@pmfst.hr
Abbreviations: APase, Alkaline phosphatase from E coli; APaseP i ,
Alkaline phosphatase from E coli containing inorganic phosphate;
2A2M1P, 2-amino-2-methyl-1-propanol; pNP, p-nitrophenol;
p-NPP, p-nitrophenyl phosphate hexahydrate disodium salt.
Enzymes: Alkaline phosphatase (PPB ECOLI, P00634),
(E.C 3.1.3.1.).
(Received 2 July 2003, revised 4 September 2003,
accepted 10 September 2003)
Trang 2with the flip-flop and half-of-the-sites mechanism [18] In
our previous work, APase from E coli displayed deviations
from Michaelis–Menten kinetics, producing concave
(downwards) Hanes plots [19], the effect being more
pronounced in the presence of a competitive inhibitor
Non-linear regression fitting, applied to equations
descri-bing models based on either negative cooperative
inter-actions between subunits or independent nonequivalent
active sites, revealed that deviations in the presence of a
competitive inhibitor could only be supported by a model
assuming inherently nonequivalent subunits The complex
cooperative mode of metal-ion binding, resulting in unequal
saturation of monomers with Mg2+, could lead to an in vivo
dimer asymmetry Therefore, the mode of activation with
metal ions, as well as the dependence of the kinetic
parameters and deviations from Michaelis–Menten kinetics
on the Zn2+ and Mg2+ ion concentration, have been
examined APase could be used as a model enzyme to
investigate the potential evolutionary advantage of
homo-dimeric enzymes, having such kinetic properties, over a
monomeric species Here we present a model that describes
the catalytic cycle of APase emphasizing the advantages that
such a mechanism could have in conjunction to the
proposed biological role of APase
Materials and methods
Dialysis of the enzyme preparation
APase from E coli type III-S (Sigma Chemie GmbH,
Taufkirchen, Germany) was dialyzed against three changes
of 50 mM Tris/HCl (pH 8) containing 20 mM EDTA,
followed by five changes of the same buffer without EDTA
Following dialysis, the protein concentration was
deter-mined from the absorbance at 280 nm, using an absorption
coefficient of e¼ 0.72M )1Æcm)1[20]
Metal free solutions
The Zn2+ion concentration (2.7· 10)7 and 5· 10)7M)
determined in distilled water and in
2-amino-2-methyl-1-propanol (2A2M1P) buffer, respectively, was high enough
to completely saturate all zinc binding sites in APase In
order to render the reaction mixture completely devoid of
divalent metal ions, all solutions were prepared using
distilled and deionized water, previously treated with an ion
exchange resin (Chelex 100, Sigma, St Louis, USA) with
high specific affinity for divalent metal ions Glassware was
soaked prior to use in a mixture of H2SO4and HNO3(1 : 1,
v/v), followed by washing in metal-free water Chelex 100
was added to each buffer prior to pH adjustment Enzyme
activity, determined in metal-free reaction mixtures,
com-prised 2–4% of the activity measured in the presence of
sufficient Zn2+
Incubation in the presence of metal ions
The enzyme solution was prepared by adding 15 lL of
dialyzed enzyme to 750 lL of 50 mMTris/HCl (pH 9) A
ZnSO4 and MgSO4 solution (50 lL), of an appropriate
concentration, was added to 51 lL of the enzyme
solution Prior to measurement, the incubation mixture
was placed for 23 h at 4C, followed by 1 h at room temperature
Spectrophotometric determination of the reaction rate The enzymatic activity was determined by measuring the absorbance change at k 405 nm and 25C, due to an increasing concentration of the reaction product, p-nitro-phenol (pNP), using the Lambda 40 Bio spectrophotometer (Perkin Elmer, Norwalk, USA) Activity was measured in a reaction mixture containing 2 mL of 0.35M 2A2M1P buffer (pH 10.5), 50 lL of the enzyme solution and 50 lL
of the substrate solution (p-nitrophenyl phosphate hexa-hydrate, disodium salt; pNPP) of an appropriate concen-tration in metal-free water Kinetic analysis was performed using pNPP as substrate at concentrations ranging from 0.01 to 2 mM Enzyme activation with Zn2+and Mg2+was followed using 2 mMpNPP All reaction rate measurements were performed in duplicate
Curve-fitting procedure The kinetic parameters providing the best fit to the experimental data were determined using the nonlinear regression data analysis program,GRAFIT, and the Hanes transformation of the equation developed for a model of
an asymmetric enzyme [19] Curves and kinetic constants, describing competitive inhibition, were obtained from respective data by applying the corresponding equation for competitive inhibition, using the kinetic parameters obtained without inhibitor as constants The kinetic parameters are presented in Tables 1–5 along with the standard errors obtained by nonlinear regression analysis The linearized transformation was applied, as the observed deviations from Michaelis–Menten kinetics were not readily detectable in the velocity vs substrate concentration plot
Table 2 The affinity of subunit 1 and 2 for P i in dependence of the Zn2+
to dimer ratio.
Zn2+to dimer ratio K I1 (m M ) K I2 (m M )
Table 1 The dependence of the kinetic parameters for APase from
E coli on the Zn2+to dimer ratio.
Zn 2+ to dimer ratio K S1 (m M ) K S2 (m M )
V m
(lmolÆmin)1) b 1.2 : 1 0.07 ± 0.02 1.76 ± 1.25 0.92 ± 0.14 1.02 ± 0.17 1.6 : 1 0.07 ± 0.02 1.21 ± 0.45 1.16 ± 0.27 1.63 ± 0.35
2 : 1 0.08 ± 0.01 1.72 ± 0.78 1.80 ± 0.22 1.41 ± 0.17 3.6 : 1 0.03 ± 0.01 1.57 ± 0.05 1.95 ± 0.02 1.54 ± 0.90
4 : 1 0.04 ± 0.04 1.96 ± 0.62 1.90 ± 0.42 1.79 ± 1.34
Trang 3Mode of metal ion activation, and the dependence
of APase activity on the metal ion concentration
In order to clarify the mode of APase activation by Zn2+,
and to establish the appropriate Zn2+ ion concentration
in kinetic and Mg2+-activation experiments in 2A2M1P
buffer at pH 10.5, enzymatic activity was determined at a
Zn2+to dimer ratio ranging from 1 : 1 to 10 : 1 Figure 1
shows the dependence of the reaction rate on the Zn2+to
dimer ratio
Enzymatic activity increases from 0.32, in the absence of
Zn2+, to 7.26 lmol pNPÆmin)1in the presence of six Zn2+
ions per dimer A further increase of the Zn2+ ion
concentration to a Zn2+to dimer ratio of 8 : 1 and 10 : 1
reduces the enzymatic activity slightly As the M3 site of
native APase binds Mg2+[21], APase activation with Zn2+
has also been followed in the presence of 2.1· 10)5M
Mg2+ (Fig 1) In the presence of Mg2+, a maximum
activity of 9.05 lmolÆmin)1pNP was attained at a Zn2+to
dimer ratio of 4 : 1 A higher Zn2+to dimer ratio resulted in
lower activity
The presence of Mg2+increases the catalytic efficiency of
APase, although it appears that Mg2+ is not directly
involved in the catalytic step The mechanism of APase
activation by Mg2+is not fully understood The influence of
Mg2+could be limited to the subunit it binds to, or it could
act on both subunits affecting the allosteric interactions and
cooperativity that possibly exist between the subunits Both phosphate-binding and calorimetric studies suggested posi-tive cooperativity of Zn2+binding to the M1 sites of the dimeric APase [5] NMR studies indicate that metal ion migration from the M1 site of an inactive subunit to the M2 site of an active subunit is taking place [20,22] The third and the fourth Zn2+probably do not bind to APase with the same affinity, whereas Mg2+ binds to the M3 site with negative cooperativity [4–6,23] Consequently, in the pres-ence of the substrate and Zn2+ions at a Zn2+to dimer ratio
of 2 : 1, both ions bind to the same subunit, generating a dimer with only one active subunit Therefore, Mg2+ activation was studied using an enzyme fully saturated with
Zn2+and having both subunits active, and an enzyme with two Zn2+ ions bound to the dimer generating only one active subunit (Fig 2)
Table 4 The affinity of subunit 1 and 2 for P i in dependence of the
Mg2+concentration at a Zn2+to dimer ratio of 2 : 1.
[Mg 2+ ] ( M ) K I1 (m M ) K I2 (m M )
Table 5 The dependence of the kinetic parameters for APase from
E coli on the Mg2+concentration at a Zn2+to dimer ratio of 4 : 1.
[Mg 2+ ]
( M )
K S1
(m M )
K S2
(m M )
V m
(lmolÆmin)1) b – 0.04 ± 0.02 2.40 ± 2.63 1.90 ± 0.79 1.89 ± 0.66
2.1 · 10)6 0.07 ± 0.02 0.64 ± 0.26 4.95 ± 1.30 1.19 ± 0.51
2.1 · 10)5 0.07 ± 0.03 1.74 ± 1.88 5.58 ± 1.74 1.42 ± 0.42
2.1 · 10)3 0.07 ± 0.01 1.18 ± 0.43 5.10 ± 0.71 1.16 ± 0.22
Table 3 The dependence of the kinetic parameters for APase from
E coli on the Mg 2+
concentration at a Zn2+to dimer ratio of 2 : 1.
[Mg2+]
( M )
K S1
(m M )
K S2
(m M )
V m
(lmolÆmin)1) b – 0.08 ± 0.01 1.72 ± 0.78 1.47 ± 0.18 1.41 ± 0.17
2.1 · 10)6 0.07 ± 0.01 2.56 ± 3.31 2.75 ± 0.41 1.11 ± 0.70
2.1 · 10)5 0.08 ± 0.01 2.03 ± 0.53 3.05 ± 0.17 1.18 ± 0.07
2.1 · 10)3 0.08 ± 0.02 2.51 ± 1.60 3.95 ± 0.54 1.52 ± 0.23
Fig 1 Catalytic activity of APase from E coli upon reactivation with
Zn2+ The dialyzed enzyme was reactivated with Zn2+at varying
Zn 2+ to dimer ratios in Tris/HCl (pH 9) in the absence of Mg 2+ (s), and in the presence of 2.1 · 10)5M Mg2+(h) Activity was deter-mined in 0.35 M 2A2M1P buffer, pH 10.5, at 25 C using 2 m M pNPP
as substrate.
Fig 2 Semi-logarithmic plot of APase activity in dependence of the
Mg 2+ concentration The dialyzed enzyme was reconstituted with
Zn2+in Tris/HCl (pH 9) at a Zn2+to dimer ratio of 2 : 1 (h), and
4 : 1 (s) The enzymatic activity was determined at varying Mg 2+
concentration in 0.35 M 2A2M1P buffer (pH 10.5) at 25 C using
2 m pNPP as substrate.
Trang 4Although Mg2+ activates both Zn2+2APase and
Zn2+4APase, the shape of the titration curve is
fundament-ally different The lowest Mg2+ concentration used
(0.001 mM) almost completely activates Zn2+4APase, in
contrast to the stepwise process of Zn2+2APase activation,
demanding a significantly higher concentration of Mg2+
(2.1 mM) In the presence of a higher Mg2+concentration,
the Zn2+2APase activity decreases sharply A somewhat
higher Mg2+ concentration (over 4.2 mM) causes the
activity to drop for the Zn2+4APase enzyme
Influence of Zn2+on the kinetic parameters
and the deviations from Michaelis–Menten kinetics
A vast amount of data indicates that the subunits of the
homodimeric APase from E coli often do not display equal
kinetic properties It has been determined that Pibinds to
APase with negative cooperativity [6,8,9,24,25], the thermal
inactivation has biphasic kinetics [26], and curve-fitting
indicates that the deviations from Michaelis–Menten
kine-tics are the consequence of unequal kinetic properties of the
subunits [19] It is possible that negative cooperativity in
metal ion binding to the M3 site results in homodimer
asymmetry Consequently, the influence of Mg2+and Zn2+
on the kinetic properties of APase and the deviations from
Michaelis–Menten kinetics have been investigated The
kinetic properties have been determined for an enzyme
reconstituted with an increasing Zn2+to dimer ratio in the
absence (Fig 3A), and in the presence of 0.05 mM Pi
(Fig 3B)
Deviations, present over the entire range of Zn2+
concentrations examined, are apparently most pronounced
at lower values The kinetic constants, obtained using the
curve-fitting procedure and describing the affinity of the
subunits for the substrate (KS1and KS2) and for Pi(KI1and
KI2), presented in Table 1 and Table 2, respectively, are
independent of the Zn2+ion concentration In order to
support the conclusion that kinetic constants do not depend
on the Zn2+ concentration, curve-fitting was performed
with a single constant value for each parameter (an average
value for each kinetic constant was used) allowing only
different Vmvalues There was no systematic deviation of
the fit confirming that kinetic constants do not depend on
the Zn2+concentration (results not shown)
An increased Zn2+concentration results in higher Vm
values, while parameter b (determining the difference in the
concentration and/or kcatof the subunits accommodating
different conformations), does not change significantly in
dependence of the Zn2+concentration
Influence of Mg2+on the kinetic properties of APase
fromE coli
Magnesium binds to the M3 site of native APase [1] It
activates the enzyme, but does not participate directly in
phosphomonoester hydrolysis [3,4] In the presence of
Mg2+, the enzyme displays a higher Vmat a constant Km
value [6] Due to negative cooperativity in metal ion binding
to the M3 site, unequal saturation of the subunits with
Mg2+ could be the principal cause of conformational
asymmetry of the homodimeric enzyme Reaction mixtures
with and without 0.05 mMP, at a Zn2+to dimer ratio of
2 : 1 (Fig 4A,B) and 4 : 1 (Fig 5A,B), have been supple-mented with 2.1· 10)6, 2.1· 10)5 and 2.1· 10)3 M
Mg2+ Deviations from linearity in the Hanes plot occur at all
Mg2+concentrations examined Deviations are apparently reduced in the presence of higher Zn2+ and Mg2+ concentrations, yet curve-fitting provides kinetic constants (KS1, KS2, b, KI1and KI2), presented in Tables 3–6, that do not differ significantly for the metal ion concentrations tested That conclusion was confirmed by successive curve-fitting with a single constant value for each parameter claimed to be independent of the Zn2+concentration (an average of all values determined for each experiment was used) allowing only Vmto change (results not shown) Upon addition of Mg2+, Vm gradually increases in reaction mixtures containing a lower Zn2+ to dimer ratio In the presence of a higher Zn2+to dimer ratio, Vmapproaches the maximum value even at the lowest Mg2+concentration tested Increasing Zn2+and Mg2+concentrations do not affect the difference between the subunits with respect for their affinity for the substrate or the product (the difference
Fig 3 The influence of Zn 2+ on the kinetic properties of APase from
E coli Catalytic activity was measured in 2A2M1P buffer, (pH 10.5)
at 25 C in the absence of P i (A) and in the presence of 0.05 m M P i (B)
at a Zn 2+ to dimer ratio of 1.2 : 1 (.), 1.6 : 1 (n), 2 : 1 (d), 3.6 : 1 (s), and 4 : 1 (+).
Trang 5between KS1and KS2, and KI1and KI2, respectively) Also,
parameter b is not significantly dependent on the metal ion
concentration It is noteworthy that the subunit with the
highest affinity for the substrate almost has the same affinity
for the product (the KI1values are only slightly lower than
the KS1values), while the subunit with the lowest affinity for
the substrate could bind Pimore tightly (KI2is considerably
lower than KS2)
Discussion
Activation with Zn2+
Maximum activity, achieved at a Zn2+to dimer ratio of
6 : 1 in the absence of Mg2+, is obtained when Zn2+is
bound to the M1 and M2 site on both subunits and perhaps
to one M3 site, that additionally activates the enzyme An
increased Zn2+ ion concentration reduces the enzymatic
activity indicating that binding of the last Zn2+ ion,
probably to the second M3 site, cannot supplement the role of magnesium in the kinetic mechanism In the presence
of Mg2+, maximum activity is accomplished at a Zn2+to dimer ratio of 4 : 1, probably resembling the form of the enzyme obtained with four Zn2+and one or two Mg2+ions bound [1,4] Higher Zn2+ concentrations decrease the enzymatic activity, probably by Zn2+ binding to the magnesium binding site M3 [4]
Fig 4 The influence of Mg2+on the kinetic properties of APase from
E coli The influence of Mg2+on the kinetic properties of APase from
E coli in the presence of a Zn2+to dimer ratio of 2 : 1 in 2A2M1P
buffer, (pH 10.5) at 25 C in the absence of P i (A), and in the presence
of 0.05 m M P i (B) The reaction was followed in reaction mixtures
containing either no Mg2+(+), or 2.1 · 10)6M , (s); 2.1 · 10)5M ,
(d) and 2.1 · 10)3M ( · ) Mg 2+
Fig 5 The influence of Mg2+on the kinetic properties of APase from
E coli The influence of Mg 2+ on the kinetic properties of APase from E coli at a Zn2+ to dimer ratio of 4 : 1 in 2A2M1P buffer (pH 10.5) at 25 C in the absence of P i (A), and in the presence of 0.05 m M P i (B) The reaction was followed in reaction mixtures containing either no Mg2+, (+) or 2.1 · 10)6M , (s); 2.1 · 10)5M , (d) and 2.1 · 10)3M (·) Mg 2+
Table 6 The affinity of subunit 1 and 2 for P i in dependence of the
Mg2+concentration at a Zn2+to dimer ratio of 4 : 1.
[Mg 2+ ] ( M ) K I1 (m M ) K I2 (m M )
Trang 6Activation with Mg2+
The Mg2+-dependence of APase activity was examined
with an enzyme reconstituted with Zn2+ ions at Zn2+
to dimer ratios of 2 : 1 and 4 : 1 As the activation
experiments produced curves with fundamentally different
shapes, it could be concluded that in the two reaction
mixtures APase occurs in a different form At a Zn2+to
dimer ratio of 2 : 1, due to positive cooperativity in Zn2+
binding [5] and migration of a metal ion from the M1 site
of the inactive subunit to the M2 site of an active subunit
[20,22], the enzyme is expected to be present in the form
containing two Zn2+ ions on the same monomer Also,
the different shapes of the curves indicate that the mode
of Mg2+ activation is not the same for Zn2+2APase as
for Zn2+4APase The more pronounced activity increase
with Zn2+4APase is probably due to the influence of
Mg2+ in an allosteric interaction A higher Mg2+
concentration is necessary for a successive activation of
Zn2+2APase, because the dimer with only one active
subunit cannot display allosteric interactions Hence, a
slow activation could result from the generation of an
enzyme with Zn2+at both M1 sites and Mg2+in the M2
site characterised by almost normal transphosphorylating
activity but considerably lower hydrolytic activity [9,27]
Lower Zn2+2APase and Zn2+4APase activity, in the
presence of a high Mg2+concentration, is probably due
to Mg2+binding to the zinc binding sites (M2 and M1)
It appears that in contrast to the binding of Zn2+to the
second M3 site, Mg2+ binding in the range of Mg2+
concentrations examined (if it binds at all due to negative
cooperativity) does not reduce the enzymatic activity
Deviation from linearity in the dependence
on the Zn2+ion concentration
Deviations from linearity will depend on the difference
between the subunits in their affinity for the substrate
(difference between KS1 and KS2), and on parameter b
describing the difference in Vm between the subunits
Deviations will be more pronounced if parameter b is large
and if the subunit affinities differ widely An increase in the
Zn2+concentration is followed only by an increase in Vm
with the remaining kinetic parameters not changing
con-siderably According to the kinetic parameters, deviations
from Michaelis–Menten kinetics are not reduced in the
presence of higher Zn2+concentrations In the Hanes plot,
deviations are apparently reduced as an increased Vm
reduces the slope of the curve, making the deviations less
obvious Analysis was performed by normalization of all
curves to the same Vm to verify that deviations did not
depend on the Zn2+ concentration as judged from the
kinetic constants The curves normalized by Vm were
superimposable with equally obvious deviations for all
Zn2+concentrations (results not shown) Deviations from
Michaelis–Menten kinetics were observed in the presence of
low Zn2+concentrations that cannot generate a fully
metal-saturated dimer This implies that interactions between the
subunits are not responsible for the observed deviation
Therefore, the cause of non-Michaelis–Menten kinetics
could only be due to a mixture of subunits differing in
conformation and catalytic properties Parameter b does
not change depending on the Zn2+concentration, thus, indicating that Zn2+ does not influence the equilibrium concentration of the subunits
Deviation from linearity in the dependence
of the Mg2+ion concentration
It has been determined that the affinity of the subunits for the substrate and the product does not depend on the
Mg2+concentration Curves normalized to the same Vm show the same deviations for all Mg2+ concentrations employed (results not shown) An increased Mg2+ concen-tration gradually activates the enzyme when partially saturated with Zn2+, while the fully saturated enzyme almost instantaneously achieves maximum activity at the lowest Mg2+concentration tested Such a mode of activa-tion suggests that Mg2+facilitates allosteric interactions in
an enzyme with four Zn2+ions bound Parameter b does not show any regular dependence on the Mg2+ concentra-tion Had negative cooperativity in Mg2+binding induced the dimer asymmetry, deviation from linearity would have been most pronounced in the presence of an Mg2+ concentration that saturates only one subunit As deviations are present in the reaction mixture devoid of Mg2+, it could
be concluded that Mg2+does not induce APase asymmetry Parameter b does not depend on the Mg2+concentration, indicating that Mg2+ equally enhances catalysis of both subunits
Model representation of the catalytic cycle for APase fromE coli
A model describing the catalytic mechanism of APase from E coli, based on the results of the kinetic experiments and in accordance with the data available in the literature, has been proposed The model encompasses the experi-mental data indicating dimer asymmetry [19,26], unequal affinity of subunits for Mg2+and Pi[6,9,20,24,25,28–31], conformational changes in the catalytic cycle [8,30,32–34], and the role of Mg2+ in an allosteric activation Asym-metry is an intrinsic characteristic of dimeric APase, and it
is not the consequence of unequal saturation with Mg2+ The difference in stability of the conformationally different subunits is apparently not large, allowing for the existence
of a conformationally heterogeneous mixture of subunits even in the presence of the Zn2+ ion concentration saturating only one monomer The homodimer could become asymmetric because of negative cooperativity in ligand binding The respective ligand can be an amino acid side-chain from the active site region, leading to homo-dimer asymmetry It has been established that Ser102, the amino acid acting as a primary nucleophile in the active site of APase from E coli, could adopt two conformations
in a dimer saturated with Pi [10] The proposed model (Scheme 1) assumes that subunit 1 displays high affinity for both the substrate and the product, while subunit 2 binds the ligand with considerably lower affinity Because
of a high affinity for the product, subunit 1 has a low kcat,
in contrast to subunit 2 showing a lower affinity for the product and consequently a higher kcat In the presence of
a low substrate concentration, subunit 1 is predomin-antly active (reaction path A) An increased substrate
Trang 7concentration activates the second subunit following
reaction path B and C
In the presence of a low substrate concentration,
phos-phomonoester hydrolysis proceeds via reaction path A The
high affinity subunit 1 binds the substrate molecule and a
covalent intermediate is formed accompanied by alcohol
dissociation Upon hydrolysis, Pislowly dissociates from the
high affinity subunit Higher substrate concentrations
activate reaction path B and C The APase dimer, with Pi
bound to the high affinity subunit, binds the substrate
molecule to the low affinity subunit In reaction path B, all
reactions take place on the subunit with lower affinity, while
in reaction path C, the first event is the interchange of
subunit conformations After a conformational change, Pi
dissociates easily from the low affinity subunit, leaving the
substrate tightly bound to the high affinity subunit
Reaction path B describes a mechanism with subunit 2
completely independent of subunit 1, with no
conforma-tional changes taking place in the course of the catalytic
cycle Substrate binding to subunit 2 could be followed by
a conformational change transforming dimer 12 into 21,
as described in reaction path C The kinetic constants KI1
and KI2, describing the affinity for Pi, differ less than
constants KS1 and KS2 Therefore, the dimer with the
substrate bound to the high affinity subunit (21) is more
stable than the dimer with the product bound to the
subunit with higher affinity (12) It facilitates product
release, and prevents substrate dissociation Following the
conformational change, the product could easily dissociate
from subunit 2, while the substrate remains bound to
subunit 1 for a new catalytic cycle The constants K , K
and Vmdescribe reaction path A with one active subunit, while constants KS2, KI2 and b describe the kinetic properties of paths B and C with both subunits active The advantage of an asymmetric dimer, over a mono-meric species, would be the additional possibility of enhanced or conformationally controlled product release The crystal structure and the reaction mechanism of APase from E coli, suggested by Kim and Wyckoff [1], as well as the high resolution crystal structure determined by Stec et al [10], offers clarification of the subunit affinity differences at a molecular level The crystal structure determined in the presence of Pi indicates that both substrate (phosphomonoester) and product (Pi) bind in the same way to the active site [ 1] Therefore, the enzyme with high affinity for the substrate also has a high affinity for the product The reaction product, Pi, is probably bound with even higher affinity, due to the influence of
Zn2+in the M2 site It is known that the enzyme is more easily phosphorylated with a phosphomonoester than with
Pi, and that the product of the transphosphorylation reaction dissociates much faster than Pi[9,27] It has been suggested that a possible reason for such a difference may
be the binding of Pias a trianion [9] Perhaps the trianion cannot be avoided because its generation is enhanced by the same catalytic Zn2+ion involved in the formation of the nucleophile for the hydrolysis of the covalent inter-mediate Alternatively, the mechanism that includes the trianion may have evolved in order to control the dissociation of the valuable product, Pi Therefore, some kind of a mechanism must have evolved either to prevent trianion formation, or to utilize it as a kinetic switch for controlled product release
It is probable that the active site adopts a new conforma-tion in order to separate Pifrom Zn2+occupying the M2 site The APasePiconformation, described by Stec et al [10], with a Zn2+replacing Mg2+in the M3 site and the side-chain of Ser102 removed from the phosphate binding site, could represent the conformation of the subunit allowing product dissociation As the side-chain of Ser102 is hydro-gen bonded to Thr155 at an increased distance from the catalytic Zn2+ion, this conformation could not be effective
in phosphomonoester hydrolysis If the crystal structure determined by Stec et al [10] resembles the conformation of subunit 2, reaction path B is not possible APase could catalyze phosphomonoester hydrolysis with a high kcatbut only via reaction path C that involves a conformational change from a 12- to a 21-dimer As an altered Ser102 conformation does not necessarily change the affinity for the substrate or the product, it is likely that the altered geometry
of an active site prevents formation of a trianon
The reaction velocity should depend on the frequency of the conformational change from 12 to 21, which will depend
on the concentration of the substrate inducing such a change The same conformational change could be induced
or enhanced by any ligand with a different binding affinity for subunits 1 and 2 If the ligand concentration is higher than that of the substrate, the conformational change occurs more often, enhancing the overall reaction velocity The catalytic path A, active in the presence of low substrate concentrations, could be enhanced in the same way Both activation of APase with Mg2+and kinetic data indicate that Mg2+enhances the reaction rate influencing allosteric
Scheme 1 The reaction cycle of APase from E coli High affinity
subunit 1 (h); low affinity subunit 2 (s); covalently bound inorganic
phosphate (-P); phosphomonoester (ROP); alcohol (ROH).
Trang 8interactions in the reaction mechanism of APase from
E coli It has been established that Mg2+binds to APase
with negative cooperativity [6,21] It increases the reaction
rate, while it does not affect the affinity for the substrate
According to the crystal structure, the subunit containing
Mg2+has a higher affinity for the substrate (corresponding
to subunit 1), and binds the substrate in a way that enables
catalysis Inorganic phosphate formed upon hydrolysis of
the covalent intermediate, remains bound to subunit 1 until
subunit 2 binds the substrate or Mg2+(Scheme 2)
The subunit with higher affinity for Pi has a higher
affinity for Mg2+ also Mg2+ binds to the low affinity
subunit enhancing the conformational change in path C,
and enabling a conformational change in path A, thereby
increasing the rate of both cycles
In reaction path A, binding of Mg2+to subunit 2 induces
a conformational change from 12 to 21 Inorganic
phos-phate and Mg2+dissociate from the low affinity subunit,
while the neighboring high affinity subunit can easily bind
another substrate molecule In reaction path C, the second
Mg2+ binds to subunit 2 following substrate binding It
enhances a conformational change inducing the release of
the product and Mg2+, thereby leaving an Mg2+ion and a
molecule of the substrate bound to the subunit capable of
catalyzing hydrolysis Therefore, binding of Mg2+ in a
negatively cooperative fashion to the M3 site of dimeric
APase increases the rate of the conformational change
responsible for the activation of the enzyme
Conforma-tionally controlled product dissociation could enhance
metabolite transfer to another protein as the conformational
change could be facilitated by an interaction with an acceptor protein or a transmembrane channel In case of APase it would allow simultaneous diffusion of Mg2+and
Piinto the cell It has been shown that the PiT transport system for Piin E coli cotransports Piand Mg2+[35]
Acknowledgements
This work was supported by a grant from the Croatian Ministry of Science and Technology Nr 177050.
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