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

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.

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,

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

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-

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

-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

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

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

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+

(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+

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

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

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+

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

(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

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

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

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

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+

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

, 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.

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

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

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.
References
1. Kim, E.E. & Wyckoff, H.W. (1990) Structure of alkaline phos-
phatases. Clin. Chim. Acta. 186, 175–187.
2. Anderson, R.A. & Vallee, B.L. (1975) Cobalt (III), a probe of
metal binding sites of Escherichia coli alkaline phosphatase. Proc.
NatlAcad.Sci.USA72, 394–397.

Mechanistic studies on equine liver alcohol dehydrogenase. I. The
stoichiometry relationship of the coenzyme binding sites to
the catalytic sites active in the reduction of aromatic aldehydes in
the transient state. Biochemistry 9, 185–192.
12. Lazdunski, M., Petitclerc, C., Chappelet, D. & Lazdunski, C.
(1971) Flip-flop mechanisms in enzymology. A model: the alkaline
phosphatase of Escherichia coli. Eur. J. Biochem. 20, 124–139.
13. Levitzki, A., Stallcup, W.B. & Koshland, D.E. Jr (1971) Half-
of-the-sites reactivity and the conformational states of cytidine
triphosphate synthetase. Biochemistry 10, 3371–3378.
14. Stallcup, W.B. & Koshland, D.E. Jr (1973) Half-of-the sites
reactivity and negative co-operativity: the case of yeast glycer-
aldehyde 3-phosphate dehydrogenase. J. Mol. Biol. 80, 41–62.
15. Stallcup, W.B. & Koshland, D.E. Jr (1973) Half-of-the sites
reactivity in the catalytic mechanism of yeast glyceraldehyde
3-phosphate dehydrogenase. J. Mol. Biol. 80, 77–91.
16. Ward, W.H. & Fersht, A.R. (1988) Tyrosyl-tRNA synthetase acts
as an asymmetric dimer in charging tRNA. A rationale for half-
of-the-sites activity. Biochemistry 27, 5525–5530.
17. Ward, W.H. & Fersht, A.R. (1988) Asymmetry of tyrosyl-tRNA
synthetase in solution. Biochemistry 27, 1041–1049.
Scheme 2. The reaction cycle of APase from E. coli in the presence of
Mg
2+
. High affinity subunit 1 (h); low affinity subunit 2 (s); cova-
lently bound inorganic phosphate (-P); phosphomonoester (ROP);
alcohol (ROH).
Ó FEBS 2003 Catalytic cycle of alkaline phosphatase from E. coli (Eur. J. Biochem. 270) 4363
18. Waight, R.D., Leff, P. & Bardsley, W.G. (1977) Steady-state
kinetic studies of the negative co-operativity and flip-flop

113 nuclear magnetic resonance of the structural basis for metal
ion dependent anticooperativity in alkaline phosphatase. Bio-
chemistry 19, 4031–4043.
23. Lazdunski, C., Petitclerc, C., Chappelet, D., Leterrier, F.,
Douzou, P. & Lazdunski, M. (1970) Tight and loose metal binding
sites in the apoalkaline phosphatase of E. coli. Reconstitution of
the Ca
2+
-phosphatase from the apoenzyme EPR study of
the Mn
2+
-phosphatase. Biochem. Biophys. Res. Commun. 40,
589–593.
24. Applebury, M.L., Johnson, B.P. & Coleman, J.E. (1970) Phos-
phate binding to alkaline phosphatase. Metal ion dependence.
J. Biol. Chem. 245, 4968–4976.
25. Chappelet-Tordo, D., Iwatsubo, M. & Lazdunski, M. (1974)
Negative cooperativity and half of the sites reactivity. Alkaline
phosphatases of Escherichia coli with Zn
2+
,Co
2+
,Cd
2+
,Mn
2+
,
and Cu
2+
in the active sites. Biochemistry 13, 3754–3762.

35. van Veen, H.W. (1997) Phosphate transport in prokaryotes:
molecules, mediators and mechanisms. Antonie Van Leeuwenhoek
72, 299–315.
4364 S. Orhanovic
´
and M. Pavela-Vranc
ˇ
ic
ˇ
(Eur. J. Biochem. 270) Ó FEBS 2003