Tài liệu Báo cáo khoa học: Dimer asymmetry and the catalytic cycle of alkaline phosphatase from Escherichia coli doc

Dimer asymmetry and the catalytic cycle of alkaline phosphatase
from
Escherichia coli
Stjepan Orhanovic
´
and Maja Pavela-Vranc
ˇ
ic
ˇ
Department of Chemistry, Faculty of Natural Sciences, Mathematics and Education, University of Split, Croatia
Although alkaline phosphatase (APase) from Escherichia
coli crystallizes 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-Vranc
ˇ
ic
ˇ
M. and Flogel-Mrs
ˇ
ic
´
M. (1994)
Acta. Pharm. 44, 87–95]. The possibility, that the observed
asymmetry could be attributed to negative cooperativity in
Mg
2+
binding, has been examined. The influence of the
metal ion content on the catalytic properties of APase from

E. coli has been examined by kinetic analyses. An activation
study has indicated that Mg
2+
enhances APase activity by a
mechanism that involves interactions between subunits. The
observed deviations from Michaelis–Menten kinetics are
independent of saturation with Zn
2+
or Mg
2+
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 Mg
2+
, as binding of Mg
2+
increases the
rate of conformational change. A conformationally con-
trolled and Mg
2+
-assisted dissociation of the reaction
product (P

i
) could serve as a kinetic switch preventing loss of
P
i
into 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
Zn
2+
-containing APase [3,4]. Significant cooperative inter-
actions have been detected during metal-ion binding,
positive for the binding of Zn
2+
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, P
i
, is rate limiting at alkaline pH.

InthecaseofP
i
hydrolysis, 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 P
i
formation is unchanged,
indicating that the newly generated phosphomonoester
dissociates much faster than P
i
. It has been suggested that
P
i
is bound to the active site in form of a dianion [9],
however, the slow dissociation of P
i
,andtheslow
phosphorylation of Ser102 by P
i
, are both in accordance
with P
i
binding 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 Mg
2+
binding site is not close enough to allow

for the direct participation of Mg
2+
in phosphomonoester
hydrolysis [9]. The crystal structure of APase in complex
with P
i
(APaseP
i
), 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 APaseP
i
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 Ôhalf-of-the-sitesÕ 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-Vranc
ˇ
ic
ˇ
, 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:
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)
Eur. J. Biochem. 270, 4356–4364 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03829.x
with 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 Mg
2+
, 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 Zn
2+
and Mg
2+
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 m
M
Tris/HCl (pH 8) containing 20 m
M
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 e ¼ 0.72
M
)1

Æcm
)1
[20].
Metal free solutions
The Zn
2+
ion concentration (2.7 · 10
)7
and 5 · 10
)7
M
)
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 H
2
SO
4
and HNO
3
(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 Zn
2+
.
Incubation in the presence of metal ions
The enzyme solution was prepared by adding 15 lLof
dialyzed enzyme to 750 lLof50m
M
Tris/HCl (pH 9). A
ZnSO
4
and MgSO
4
solution (50 lL), of an appropriate
concentration, was added to 51 lLoftheenzyme
solution. Prior to measurement, the incubation mixture
was placed for 23 h at 4 °C, 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 25 °C, 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 0.35
M
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 m
M
. Enzyme activation with Zn
2+
and Mg
2+
was
followed using 2 m
M
pNPP. 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
,andtheHanes
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 Zn
2+
to dimer ratio.
Zn
2+
to dimer ratio K
I1
(m
M
) K
I2
(m
M
)
1.2 : 1 0.04 ± 0.004 0.19 ± 0.04
1.6 : 1 0.03 ± 0.004 0.12 ± 0.02
2 : 1 0.04 ± 0.004 0.27 ± 0.07
3.6 : 1 0.02 ± 0.003 0.13 ± 0.02
4 : 1 0.03 ± 0.01 0.10 ± 0.04
Table 1. The dependence of the kinetic parameters for APase from
E. coli on the Zn
2+
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
Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4357
Results
Mode 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 Zn
2+
,
and to establish the appropriate Zn
2+
ion concentration
in kinetic and Mg
2+

-activation experiments in 2A2M1P
buffer at pH 10.5, enzymatic activity was determined at a
Zn
2+
to dimer ratio ranging from 1 : 1 to 10 : 1. Figure 1
shows the dependence of the reaction rate on the Zn
2+
to
dimer ratio.
Enzymatic activity increases from 0.32, in the absence of
Zn
2+
,to7.26lmol pNPÆmin
)1
in the presence of six Zn
2+
ions per dimer. A further increase of the Zn
2+
ion
concentration to a Zn
2+
to dimer ratio of 8 : 1 and 10 : 1
reduces the enzymatic activity slightly. As the M3 site of
native APase binds Mg
2+
[21], APase activation with Zn
2+
has also been followed in the presence of 2.1 · 10
)5
M

Mg
2+
(Fig. 1). In the presence of Mg
2+
, a maximum
activity of 9.05 lmolÆmin
)1
pNP was attained at a Zn
2+
to
dimer ratio of 4 : 1. A higher Zn
2+
to dimer ratio resulted in
lower activity.
The presence of Mg
2+
increases the catalytic efficiency of
APase, although it appears that Mg
2+
is not directly
involved in the catalytic step. The mechanism of APase
activation by Mg
2+
is not fully understood. The influence of
Mg
2+
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 Zn
2+
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 Zn
2+
probably do not bind to APase with the
same affinity, whereas Mg
2+
binds to the M3 site with
negative cooperativity [4–6,23]. Consequently, in the pres-
ence of the substrate and Zn
2+
ions at a Zn
2+
to dimer ratio
of 2 : 1, both ions bind to the same subunit, generating a
dimer with only one active subunit. Therefore, Mg
2+
activation was studied using an enzyme fully saturated with
Zn
2+
and having both subunits active, and an enzyme with
two Zn
2+
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
Mg
2+
concentration at a Zn
2+
to dimer ratio of 2 : 1.
[Mg
2+
](
M
) K
I1
(m
M
) K
I2
(m
M
)
– 0.04 ± 0.004 0.37 ± 0.10
2.1 · 10
)6
0.04 ± 0.001 0.20 ± 0.05
2.1 · 10
)5
0.04 ± 0.003 0.12 ± 0.01
2.1 · 10
)3
0.05 ± 0.003 0.74 ± 0.41

Table 5. The dependence of the kinetic parameters for APase from
E. coli on the Mg
2+
concentration at a Zn
2+
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 Zn
2+
to dimer ratio of 2 : 1.
[Mg
2+
]
(
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
Zn
2+
. The dialyzed enzyme was reactivated with Zn
2+
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
)5
M
Mg
2+

(h). Activity was deter-
minedin0.35
M
2A2M1P buffer, pH 10.5, at 25 °Cusing2m
M
pNPP
as substrate.
Fig. 2. Semi-logarithmic plot of APase activity in dependence of the
Mg
2+
concentration. Thedialyzedenzymewasreconstitutedwith
Zn
2+
in Tris/HCl (pH 9) at a Zn
2+
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 °Cusing
2m
M
pNPP as substrate.
4358 S. Orhanovic
´
and M. Pavela-Vranc
ˇ
ic
ˇ

(Eur. J. Biochem. 270) Ó FEBS 2003
Although Mg
2+
activates both Zn
2+
2
APase and
Zn
2+
4
APase, the shape of the titration curve is fundament-
ally different. The lowest Mg
2+
concentration used
(0.001 m
M
) almost completely activates Zn
2+
4
APase, in
contrast to the stepwise process of Zn
2+
2
APase activation,
demanding a significantly higher concentration of Mg
2+
(2.1 m
M
). In the presence of a higher Mg
2+

concentration,
the Zn
2+
2
APase activity decreases sharply. A somewhat
higher Mg
2+
concentration (over 4.2 m
M
)causesthe
activity to drop for the Zn
2+
4
APase enzyme.
Influence of Zn
2+
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 P
i
binds 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 Mg

2+
and Zn
2+
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
reconstitutedwithanincreasingZn
2+
to dimer ratio in the
absence (Fig. 3A), and in the presence of 0.05 m
M
P
i
(Fig. 3B).
Deviations, present over the entire range of Zn
2+
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 (K
S1
and K
S2
)andforP
i
(K
I1
and
K
I2

), presented in Table 1 and Table 2, respectively, are
independent of the Zn
2+
ion concentration. In order to
support the conclusion that kinetic constants do not depend
on the Zn
2+
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 V
m
values. There was no systematic deviation of
the fit confirming that kinetic constants do not depend on
the Zn
2+
concentration (results not shown).
An increased Zn
2+
concentration results in higher V
m
values, while parameter b (determining the difference in the
concentration and/or k
cat
of the subunits accommodating
different conformations), does not change significantly in
dependence of the Zn
2+
concentration.
Influence of Mg

2+
on the kinetic properties of APase
from
E. 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
Mg
2+
, the enzyme displays a higher V
m
at a constant K
m
value [6]. Due to negative cooperativity in metal ion binding
to the M3 site, unequal saturation of the subunits with
Mg
2+
could be the principal cause of conformational
asymmetry of the homodimeric enzyme. Reaction mixtures
with and without 0.05 m
M
P
i
,ataZn
2+
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
Mg
2+
.
Deviations from linearity in the Hanes plot occur at all
Mg
2+
concentrations examined. Deviations are apparently
reduced in the presence of higher Zn
2+
and Mg
2+
concentrations, yet curve-fitting provides kinetic constants
(K
S1
, K
S2
, b, K
I1
and K
I2
), 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 Zn
2+

concentration (an
average of all values determined for each experiment was
used) allowing only V
m
to change (results not shown). Upon
addition of Mg
2+
, V
m
gradually increases in reaction
mixtures containing a lower Zn
2+
to dimer ratio. In the
presence of a higher Zn
2+
to dimer ratio, V
m
approaches
the maximum value even at the lowest Mg
2+
concentration
tested. Increasing Zn
2+
and Mg
2+
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 °CintheabsenceofP
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 (+).
Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4359
between K
S1
and K
S2
,andK
I1
and K
I2
, 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 K
I1
values are only slightly lower than
the K

S1
values), while the subunit with the lowest affinity for
the substrate could bind P
i
more tightly (K
I2
is considerably
lower than K
S2
).
Discussion
Activation with Zn
2+
Maximum activity, achieved at a Zn
2+
to dimer ratio of
6 : 1 in the absence of Mg
2+
, is obtained when Zn
2+
is
bound to the M1 and M2 site on both subunits and perhaps
to one M3 site, that additionally activates the enzyme. An
increased Zn
2+
ion concentration reduces the enzymatic
activity indicating that binding of the last Zn
2+
ion,
probably to the second M3 site, cannot supplement the

role of magnesium in the kinetic mechanism. In the presence
of Mg
2+
, maximum activity is accomplished at a Zn
2+
to
dimer ratio of 4 : 1, probably resembling the form of the
enzyme obtained with four Zn
2+
and one or two Mg
2+
ions
bound [1,4]. Higher Zn
2+
concentrations decrease the
enzymatic activity, probably by Zn
2+
binding to the
magnesium binding site M3 [4].
Fig. 4. The influence of Mg
2+
on the kinetic properties of APase from
E. coli. The influence of Mg
2+
on the kinetic properties of APase from
E. coli inthepresenceofaZn
2+
to dimer ratio of 2 : 1 in 2A2M1P
buffer, (pH 10.5) at 25 °CintheabsenceofP
i

(A), and in the presence
of 0.05 m
M
P
i
(B). The reaction was followed in reaction mixtures
containing either no Mg
2+
(+), or 2.1 · 10
)6
M
,(s); 2.1 · 10
)5
M
,
(d)and2.1· 10
)3
M
( · )Mg
2+
.
Fig. 5. The influence of Mg
2+
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 Zn
2+
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 Mg
2+
,(+)or2.1· 10
)6
M
,(s); 2.1 · 10
)5
M
,
(d)and2.1· 10
)3
M
(·)Mg
2+
.
Table 6. The affinity of subunit 1 and 2 for P
i
in dependence of the
Mg
2+
concentration at a Zn
2+

to dimer ratio of 4 : 1.
[Mg
2+
](
M
) K
I1
(m
M
) K
I2
(m
M
)
– 0.04 ± 0.005 0.12 ± 0.04
2.1 · 10
)6
0.04 ± 0.002 0.11 ± 0.01
2.1 · 10
)5
0.05 ± 0.007 0.45 ± 0.19
2.1 · 10
)3
0.05 ± 0.008 0.42 ± 0.27
4360 S. Orhanovic
´
and M. Pavela-Vranc
ˇ
ic
ˇ

(Eur. J. Biochem. 270) Ó FEBS 2003
Activation with Mg
2+
The Mg
2+
-dependence of APase activity was examined
with an enzyme reconstituted with Zn
2+
ions at Zn
2+
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 Zn
2+
to
dimer ratio of 2 : 1, due to positive cooperativity in Zn
2+
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 Zn
2+
ions on the same monomer. Also,
the different shapes of the curves indicate that the mode
of Mg
2+
activation is not the same for Zn
2+
2

APase as
for Zn
2+
4
APase. The more pronounced activity increase
with Zn
2+
4
APase is probably due to the influence of
Mg
2+
in an allosteric interaction. A higher Mg
2+
concentration is necessary for a successive activation of
Zn
2+
2
APase, 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 Zn
2+
at both M1 sites and Mg
2+
in the M2
site characterised by almost normal transphosphorylating
activity but considerably lower hydrolytic activity [9,27].
Lower Zn
2+
2

APase and Zn
2+
4
APase activity, in the
presence of a high Mg
2+
concentration, is probably due
to Mg
2+
binding to the zinc binding sites (M2 and M1).
It appears that in contrast to the binding of Zn
2+
to the
second M3 site, Mg
2+
binding in the range of Mg
2+
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 Zn
2+
ion concentration
Deviations from linearity will depend on the difference
between the subunits in their affinity for the substrate
(difference between K
S1
and K
S2
), and on parameter b

describing the difference in V
m
between the subunits.
Deviations will be more pronounced if parameter b is large
and if the subunit affinities differ widely. An increase in the
Zn
2+
concentration is followed only by an increase in V
m
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 Zn
2+
concentrations. In the Hanes plot,
deviations are apparently reduced as an increased V
m
reduces the slope of the curve, making the deviations less
obvious. Analysis was performed by normalization of all
curves to the same V
m
to verify that deviations did not
depend on the Zn
2+
concentration as judged from the
kinetic constants. The curves normalized by V
m
were
superimposable with equally obvious deviations for all
Zn

2+
concentrations (results not shown). Deviations from
Michaelis–Menten kinetics were observed in the presence of
low Zn
2+
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 Zn
2+
concentration, thus,
indicating that Zn
2+
does not influence the equilibrium
concentration of the subunits.
Deviation from linearity in the dependence
of the Mg
2+
ion concentration
It has been determined that the affinity of the subunits
for the substrate and the product does not depend on the
Mg
2+
concentration. Curves normalized to the same V
m
show the same deviations for all Mg
2+

concentrations
employed (results not shown). An increased Mg
2+
concen-
tration gradually activates the enzyme when partially
saturated with Zn
2+
, while the fully saturated enzyme
almost instantaneously achieves maximum activity at the
lowest Mg
2+
concentration tested. Such a mode of activa-
tion suggests that Mg
2+
facilitates allosteric interactions in
an enzyme with four Zn
2+
ions bound. Parameter b does
not show any regular dependence on the Mg
2+
concentra-
tion. Had negative cooperativity in Mg
2+
binding induced
the dimer asymmetry, deviation from linearity would have
been most pronounced in the presence of an Mg
2+
concentration that saturates only one subunit. As deviations
are present in the reaction mixture devoid of Mg
2+

, it could
be concluded that Mg
2+
does not induce APase asymmetry.
Parameter b does not depend on the Mg
2+
concentration,
indicating that Mg
2+
equally enhances catalysis of both
subunits.
Model representation of the catalytic cycle
for APase from
E. 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 Mg
2+
and P
i
[6,9,20,24,25,28–31],
conformational changes in the catalytic cycle [8,30,32–34],
and the role of Mg
2+
in an allosteric activation. Asym-
metry is an intrinsic characteristic of dimeric APase, and it
is not the consequence of unequal saturation with Mg

2+
.
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 Zn
2+
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 P
i
[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 k
cat
,
in contrast to subunit 2 showing a lower affinity for the
product and consequently a higher k
cat
. In the presence of
a low substrate concentration, subunit 1 is predomin-
antly active (reaction path A). An increased substrate

Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4361
concentration 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, P
i
slowly dissociates from the
high affinity subunit. Higher substrate concentrations
activate reaction path B and C. The APase dimer, with P
i
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, P
i
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 K
I1
and K
I2

, describing the affinity for P
i
,differlessthan
constants K
S1
and K
S2
. 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
S1
, K
I1
and V
m
describe reaction path A with one active subunit,
while constants K
S2
, K
I2
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 P
i
indicates that both
substrate (phosphomonoester) and product (P
i
)bindin
thesamewaytotheactivesite[1].Therefore,theenzyme
with high affinity for the substrate also has a high affinity
for the product. The reaction product, P
i
, is probably
bound with even higher affinity, due to the influence of
Zn
2+
in the M2 site. It is known that the enzyme is more
easily phosphorylated with a phosphomonoester than with
P
i
, and that the product of the transphosphorylation
reaction dissociates much faster than P
i
[9,27]. It has been
suggested that a possible reason for such a difference may
be the binding of P
i

as a trianion [9]. Perhaps the trianion
cannot be avoided because its generation is enhanced by
the same catalytic Zn
2+
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, P
i
. 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 P
i
from Zn
2+
occupying the M2
site. The APaseP
i
conformation, described by Stec et al. [10],
with a Zn
2+
replacing Mg
2+
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 Zn
2+
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 k
cat
but
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 Mg
2+
and kinetic data indicate
that Mg

2+
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).
4362 S. Orhanovic
´
and M. Pavela-Vranc
ˇ
ic
ˇ
(Eur. J. Biochem. 270) Ó FEBS 2003
interactions in the reaction mechanism of APase from
E. coli. It has been established that Mg
2+
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
Mg
2+
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 Mg
2+
(Scheme 2).
The subunit with higher affinity for P
i

has a higher
affinity for Mg
2+
also. Mg
2+
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 Mg
2+
to subunit 2 induces
a conformational change from 12 to 21. Inorganic phos-
phate and Mg
2+
dissociate from the low affinity subunit,
while the neighboring high affinity subunit can easily bind
another substrate molecule. In reaction path C, the second
Mg
2+
binds to subunit 2 following substrate binding. It
enhances a conformational change inducing the release of
the product and Mg
2+
, thereby leaving an Mg
2+
ion and a
molecule of the substrate bound to the subunit capable of
catalyzing hydrolysis. Therefore, binding of Mg
2+

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 Mg
2+
and
P
i
into the cell. It has been shown that the PiT transport
system for P
i
in E. coli cotransports P
i
and Mg
2+
[35].
Acknowledgements
This work was supported by a grant from the Croatian Ministry of
Science and Technology Nr. 177050.
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ˇ
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