The catalytic properties of alkaline phosphatases under various conditions

Article abstract of DOI:10.1134/S0036024408110265

A comparative study was performed to examine the catalytic properties of alkaline phosphatases from bacteria Escherichia coli and bovine and chicken intestines. The activity of enzyme dimers and tetramers was determined. The activity of the dimer was three or four times higher than that of the tetramer. The maximum activity and affinity for 4-nitrophenylphosphate was observed for the bacterial alkaline phosphatase (K_M = 1.7 × 10^-5 M, V_max = 1800 μmol/(min mg of protein) for dimers and V _max = 420 μmol/(min mg of protein) for tetramers). The Michaelis constants were equal for two animal phosphatases in various buffer media (pH 8.5) ((3.5 ± 0.2) × 10^-4 M). Five buffer systems were investigated: tris, carbonate, hepes, borate, and glycine buffers, and the lowest catalytic activity of alkaline phosphatases at equal pH was observed in the borate buffer (for enzyme from bovine intestine, V_max = 80 μmol/(min mg of protein)). Cu²⁺ cations formed a complex with tris-(oxymethyl)-aminomethane (tris-HCl buffer) and inhibited the intestine alkaline phosphatases by a noncompetitive mechanism.

Full text of DOI:10.1134/S0036024408110265

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2008, Vol. 82, No. 11, pp. 1947–1951. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © L.F. Atyaksheva, E.S. Chukhrai, O.M. Poltorak, 2008, published in Zhurnal Fizicheskoi Khimii, 2008, Vol. 82, No. 11, pp. 2164–2169.
BIOPHYSICAL
CHEMISTRY
The Catalytic Properties of Alkaline Phosphatases
under Various Conditions
L. F. Atyaksheva, E. S. Chukhrai, and O. M. Poltorak†
Faculty of Chemistry, Moscow State University, Moscow, 119899 Russia
e-mail: poltorak@phys.chem.msu.ru
Received June 26, 2007
Abstract—A comparative study was performed to examine the catalytic properties of alkaline phosphatases
from bacteria Escherichia coli and bovine and chicken intestines. The activity of enzyme dimers and tetramers
was determined. The activity of the dimer was three or four times higher than that of the tetramer. The maximum
activity and affinity for 4-nitrophenylphosphate was observed for the bacterial alkaline phosphatase (KM
=
1.7 × 10–5 M, Vmax = 1800 µmol/(min mg of protein) for dimers and Vmax = 420 µmol/(min mg of protein) for
tetramers). The Michaelis constants were equal for two animal phosphatases in various buffer media (pH 8.5)
((3.5 0.2) × 10–4 M). Five buffer systems were investigated: tris, carbonate, hepes, borate, and glycine buffers,
and the lowest catalytic activity of alkaline phosphatases at equal pH was observed in the borate buffer (for
enzyme from bovine intestine, Vmax = 80 µmol/(min mg of protein)). Cu2+ cations formed a complex with
tris-(oxymethyl)-aminomethane (tris-HCl buffer) and inhibited the intestine alkaline phosphatases by a non-
competitive mechanism.
DOI: 10.1134/S0036024408110265
INTRODUCTION
The activity of alkaline phosphatases depends on the
oligomer composition of the enzyme and its ability to
undergo association and dissociation. Since bonds
between oligomer subunits are noncovalent, oligomers
dissociate into subunits and can experience nonspecific
association into associates larger than the starting mol-
ecule. Equilibrium between oligomeric enzyme forms
is controlled by substrates, coenzymes, and allosteric
effectors. Various oligomeric structures can have differ-
ent catalytic properties, but the question of the relation
between the quaternary structure of the enzyme and its
activity remains open.
Alkaline phosphatases (EC 3.1.3.1), which catalyze
the nonspecific hydrolysis of phosphomonoethers, are
present in almost all living organisms [1]. According to
the X-ray diffraction data on several alkaline phos-
phatases [2–5], these are dimeric enzymes containing
zinc and magnesium cations in the active center. In sev-
eral works, active tetramers were isolated along with
active dimers [6–8], while the monomeric enzyme was
inactive [1, 9]. The catalytic properties of alkaline
phosphatases depend on the presence of various cations
in the reaction mixture. First, these are the Mg2+ and
Zn2+ cations, which participate in catalytic events. Mg2+
cations can also alter the activity of alkaline phos-
phatases via interactions between subunits in the
dimeric enzyme [10]. Magnesium and zinc cations can
be removed from alkaline phosphatase with enzyme
catalytic activity loss as a result of the formation of
inactive monomers [11]. Zinc cations at concentrations
higher than 10 µM promote the association of alkaline
phosphatase dimers into tetramers [6].
The aim of this work was to reveal the role played
by association–dissociation processes in the activation
and deactivation of alkaline phosphatases of different
origins and to study the effects of the buffer medium
and metal cations on these processes.
EXPERIMENTAL
Lyophilized alkaline phosphatase samples from
bacteria Esterichia coli (50% protein, Sigma), bovine
intestine (25% protein, Sigma), and chicken intestine
(12% protein, Reanal) were used. The protein content
in the samples was determined by the Bradford method.
The catalytic activity of the enzymes was estimated
from the initial rate of hydrolysis of a synthetic sub-
strate (4-nitrophenyl phosphate), whose hydrolysis
product (4-nitrophenol) was identified spectrophoto-
metrically.
Alkaline phosphatase can be activated not only by
Mg2+ and Zn2+, but also by some other cations present
in the reaction mixture (Ca2+, Mn2+, Ni2+, and Co2+) [9,
12]. It is inhibited by the Hg2+, Ag+, Bi2+, and Cu2+ cat-
ions [12]. Several divalent cations can replace Mg2+ and
Zn2+ in the active center of the enzyme with a decrease
in catalytic activity sometimes by a factor of 5000 [13].
†
Deceased.
1947

1948
ATYAKSHEVA et al.
shown in Fig. 1a, the specific activity of alkaline phos-
a, µmol/(min mg of protein)
phatases from chicken and bovine intestines and bacte-
ria E. coli in a carbonate buffer increases substantially
when the enzyme concentration decreases, the bacterial
enzyme being most active. The activity of alkaline
phosphatases also depends on the buffer system in
which the catalytic reaction is conducted. Figure 1b
shows the dependences of the specific activity of
bovine alkaline phosphatase on concentration in five
different buffer solutions at constant pH 8.5. The lowest
activity is observed in the borate buffer, in which the
specific activity remaines constant over the whole
range of enzyme concentrations (0.02–20 mg/l). The
highest specific activity, especially at low concentra-
tions, is observed in the buffer solution of tris-(oxyme-
thyl)-aminomethane–HCl. The dependence of the spe-
cific activity of alkaline phosphatases on the enzyme
concentration in solution suggests that oligomeric
enzymes undergo association–dissociation.
800
(‡)
600
400
200
3
2
1
0
0.1
0.2
0.3
(b)
0.4 2
4
[E]0, mg/l
a, µmol/(min mg of protein)
1000
According to modern views, active dimers (Ö2) are
the basic structural units of alkaline phosphatases capa-
ble of being associated into less active tetramers (Ö4)
under certain conditions. Monomers (Ö1) formed in the
dissociation of dimers have no catalytic activity. The
specific activity of the enzyme is the sum of two terms,
each of which is the product of the specific catalytic
activity of the corresponding oligomeric form by its
fraction. Thus, for the equilibrium 2Ö2 ⇔ Ö4,
800
600
400
200
5
4
3
2
1
0
0.1 0.2 0.3 0.4
3
4
5
[E2]
----------
[E4]
---------- -----------------------------------------------
= a4 +
2(a2 – a4)
[E]0, mg/l
a = a
+ a
2[E]0
4[E]0
1 + (1 + 8K[E]0)1/2
(1)
Fig. 1. Dependence of the specific activity of alkaline phos-
phatases from (a) (1) chicken intestine, (2) bovine intestine,
and (3) bacteria E. coli and (b) bovine intestine on the enzyme
concentration in (a) carbonate buffer and (b) (1) borate buffer,
(2) hepes, (3) glycine buffer, (4) carbonate buffer, and (5) tris;
pH 8.5.
(1 + 8K[E]0)1/2 – 1
-----------------------------------------------
= a2 + (a4 – a2)
,
(1 + 8K[E]0)1/2 + 1
where a2 and a4 are the specific activities of dissocia-
tion products Ö2 and enzyme associates Ö4, respec-
tively, and [E]0 is the initial concentration of the
enzyme. In very dilute solutions, the activity of associ-
ates can be ignored. Equation (1) then becomes
The specific activity of the enzyme (in µmol/(min
mg of protein) was determined over the alkaline phos-
phatase concentration range 3 × 10–3–15.0 mg/l in vari-
ous buffer media. The 0.1 M buffer systems (pH 8.5)
used included tris-(oxymethyl)aminomethane–HCl
(tris), NaHCO3–Na2CO3 (carbonate), 2-[4-(2-hydroxy-
ethyl)-1-piperazinyl]-ethane–HCl (hepes), Na4B4O7–
HCl (borate), and glycine–NaOH (glycine). The spe-
cific activities of dimers and tetramers of alkaline phos-
phatases were determined from the concentration depen-
dences of the specific activity of the enzymes [14].
2K‡ÒÒ
1
1
------------
-- = ---- +
A,
(2)
a22
a
a2
where A = a[E]0 is the total enzyme activity. Using (2),
one can determine the specific activity of dimers by
extrapolating the experimental data to zero enzyme
concentration.
Figure 2 shows the experimental data on the specific
activity of bovine alkaline phosphatase in various alka-
line solutions. The table lists the data on alkaline phos-
phatase dimers. In the tris-HCl buffer, the activity of
dimers is higher than their activity in other buffer solu-
tions. Comparing dimer activities under equal condi-
tions (carbonate buffer in our case, pH 8.5), one can see
Copper and lead acetates in concentrations of 0.1–
10 mM were used as inhibitors of alkaline phosphatase.
The type of inhibition was determined.
RESULTS AND DISCUSSION
The alkaline phosphatases under study differ in their that the activity of bacterial enzyme is higher by factors
catalytic activity with respect to the hydrolysis of of 3 and 4.5 than the activities of bovine and chicken
4-nitrophenylphosphate. According to the dependences phosphatases, respectively.
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THE CATALYTIC PROPERTIES OF ALKALINE PHOSPHATASES
1949
103/a, min mg/µmol
a, µmol/(min mg of protein)
4
4
400
2
1
3
3
2
1
300
2
1
200
100
0
20
40
60
80
0
0.02
0.04
0.06
0.08
(a2 – a/[E0])1/2, (µmol/min)1/2
V, µmol/min
Fig. 2. Determination of the specific activity of dimers in
the coordinates of Eq. (2). Alkaline phosphatase from
bovine intestine, pH 8.5, in (1) tris, (2) carbonate buffer,
(3) glycine buffer, (4) hepes.
Fig. 3. Determination of the specific activity of alkaline
phosphatase tetramers from bovine intestine in the coordi-
nates of Eq. (3) in (1) hepes and (2) carbonate buffer.
The experimental concentration dependences of the do not dissociate into inactive monomers. Equilibrium
specific activity of alkaline phosphatases also allow one can be shifted toward inactive monomers by increasing
to determine the activity of the tetrameric form of the medium acidity [9] or temperature. In all buffer sys-
enzyme. We used the approach of [14]. If the activity of tems under study except the borate buffer, tetramers are
the dimer was determined correctly using (2), the the basic structural units at enzyme concentrations
experimental data should be linearized in the coordi- above 0.5 mg/l, and equilibrium shifts toward more
nates of the equation
active dimers at concentrations lower than 0.02 mg/l. In
the borate buffer, the tetramer form does not dissociate
over the concentration range under study. Less active
higher associates can also form. Similar results were
reported in [9]; according to these data, recombinant
alkaline phosphatase exists in a phosphate buffer in
the form of active dimers and inactive monomers, but
only dimers are present in the tris buffer at the same
pH values.
1/2
1/2
a2 – a4
---------------
2K‡ÒÒ
a2 – a
-------------
[E]0
⎛
a = a4 +
⎝
⎞
⎠
⎛
⎝
⎞
⎠
.
(3)
This allows one to determine the specific activity of the
tetramer. The experimental data in the coordinates of
(3) are shown in Fig. 3. The linear dependences confirm
that the scheme of the association of alkaline phos-
phatases is correct, and this approach can be used for
determining the specific activity of the tetrameric and
dimeric enzyme forms. As to the catalytic activity of
alkaline phosphatase in the borate buffer (80 µmol/(min
mg of protein)), it can characterize the properties of tet-
ramers or higher associates.
Figure 5 shows the dependence of the activity of
alkaline phosphatases from bovine and chicken intes-
tines on the content of copper cations in a tris-HCl
buffer solution (pH 8.5). Note that, at pH 8.5 in this
range of copper cation concentrations, Cu(OH)2 precip-
itate should form (5 × 10–20), which does occur in the
carbonate buffer. Cu2+ cations presumably form a coor-
The results of this study show that, in different buff-
ers with equal pH, the specific activities of alkaline
phosphatase dimers are three or four times higher than
the activities of tetramers. The Michaelis constant
(Fig. 4) is independent of the type of the buffer system
and enzyme concentration in solution. For alkaline
phosphatases from bovine or chicken intestines, Kå =
(3.5 0.2) × 10–4 M; for bacterial phosphatase, Kå =
1.7 × 10–5 M. Thus, while having different catalytic
activities, alkaline phosphatase dimers and tetramers
have equal affinities for the synthetic substrate, which
is higher for bacterial phosphatase and lower for animal
phosphatases.
Specific activities (µmol/(min mg of protein)) of alkaline
phosphatase dimers (a2) and tetramers (a4) from bovine (I)
and chicken (II) intestines and bacteria E. coli (III) (pH 8.5)
Buffer
Phosphatase
a2
a4
Tris
I
1100
500
260
140
115
220
100
420
Glycine type
Hepes
I
I
450
Carbonate type
Carbonate type
Carbonate type
I
650
Let us consider equilibria in the Ö4 ⇔ 2Ö2 ⇔ 4Ö1
system. Under the conditions of our experiments, we do
not observe the last equilibrium; that is, active dimers
II
III
400
1800
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 82 No. 11 2008

1950
ATYAKSHEVA et al.
1/V, min mg/µmol
60
V/V0
1.0
4
3
0.8
0.6
0.4
0
1
2
3
40
20
2
1
2
4
6
[Cu2+], må
Fig. 5. Dependences of the relative activity (V/V ) of alka-
0
line phosphatases from (1, 2) bovine and (3) chicken intes-
0
5
10
15
20
2+
2+
1/[S], må–1
tines on the content of (1, 3) Cu and (2) Pb cations in
tris-HCl buffer.
Fig. 4. Determination of the Michaelis–Menten equation
parameters for alkaline phosphatases from (1–3) bovine
intestine and (4) chicken intestine in (1, 2, 4) carbonate
buffer at pH 8.5 and (3) borate buffer at pH 8.5 at enzyme
concentrations of (1) 0.025 and (2–4) 2.5 mg/l.
in the tris-HCl buffer system decreases not only the cat-
alytic activity of the enzyme, but also the Michaelis
constant (from 3.6 × 10–4 to 2.2 × 10–4 M) (Fig. 6).
A simultaneous decrease in the Michaelis constant
and maximum reaction rate (enzyme activity) corre-
sponds to the noncompetitive type of inhibition. In this
case, the alkaline phosphatase is likely inhibited by the
coordination compound of copper cations with tris-
(oxymethyl)-aminomethane rather than by copper cat-
ions. According to [15], alkaline phosphatase from calf
intestine is inhibited by complexes of hexaphosphati-
nositol and divalent metal cations (Mn2+, Ni2+, Cd2+,
and Co2+).
dination bond with the tris-(oxymethyl)-aminomethane
molecule. Cu(OH)2 forms in this buffer at copper con-
centrations higher than 12 mM. As can be seen in Fig. 6,
the activity of alkaline phosphatases decreases as the
Cu2+ concentration increases in the buffer solution. At
copper concentrations above ≈4 mM, the activity of
alkaline phosphatases decreases by a factor of two
compared with the activity in the absence of copper cat-
ions. A similar effect on the activity of alkaline phos-
phatases is produced by lead cations present in the
Alkaline phosphatase from crab is inhibited by cop-
buffer solution (Fig. 5). The presence of copper cations per cations at concentrations of 50–150 µM in a car-
1/V, mg min/µmol
35
3
30
25
20
2
15
1
10
5
–4
–2
0
2
4
6
8
1/[S], må–1
Fig. 6. Determination of the Michaelis–Menten equation parameters and the type of inhibition of alkaline phosphatase from bovine
2+
intestine by copper cations; [Cu ] = 0 (1), 2.5 (2), and 6.0 mM (3).
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 82 No. 11 2008

THE CATALYTIC PROPERTIES OF ALKALINE PHOSPHATASES
1951
source of the enzyme, but also on its oligomeric com-
position, the properties of the buffer systems in which
the enzyme reaction takes place, and the presence of
several cations in the reaction mixture.
V/V0
1
1.0
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phosphatase is inactivated with time. At a 0.5 mM con-
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RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 82 No. 11 2008