Báo cáo khóa học: How calcium inhibits the magnesium-dependent enzyme human phosphoserine phosphatase potx

How calcium inhibits the magnesium-dependent enzyme human
phosphoserine phosphatase
Yves Peeraer
1
, Anja Rabijns
1
, Jean-Franc¸ois Collet
2
, Emile Van Schaftingen
2
and Camiel De Ranter
1
1
Laboratory for Analytical Chemistry and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences, K.U. Leuven, Leuven,
Belgium;
2
Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology, Universite
´
Catholique de
Louvain, Brussels, Belgium
The structure of the Mg
2+
-dependent enzyme human
phosphoserine phosphatase (HPSP) was exploited to
examine the structural and functional role of the divalent
cation in the a ctive site of phosphatases. Most interesting is
the biochemical observation that a Ca
2+
ion inhibits the
activity of HPSP, even in the presence of added Mg
2+

.The
sixfold coordinated Mg
2+
ion present in the a ctive site of
HPSP under normal physiological conditions, was replaced
by a Ca
2+
ion by using a crystallization condition with high
concentration of CaCl
2
(0.7
M
). The r esulting HPSP struc-
ture now shows a sevenfold coordinated Ca
2+
ioninthe
active site that might e xplain the inhibitory effect of Ca
2+
on
the enzyme. Indeed, the Ca
2+
ion in the active site captures
both side-chain oxygen atoms of the catalytic Asp20 as a
ligand, while a Mg
2+
ion ligates only one oxygen atom of
this Asp residue. The bidentate character of Asp20 towards
Ca
2+
hampers the nucleophilic attack of one of the Asp20

side chain oxygen atoms on the phosphorus atom of the
substra te phosph oserine .
Keywords: calcium; HAD superfamily; magnesium-depend-
ent enzymes; phosphoserine phosphatase;
L
-serine.
Human phosphoserine phosphatase (HPSP) catalyses the
last and irreversible step of the de novo biosynthesis of
L
-serine, i.e. the hydrolysis of phosphoserine l eading to the
formation of
L
-serine and inorganic phosphate (Pi). HPSP is
a member of the haloacid dehalogenase (HAD) superfamily
of which the members are characterized by three short
conserved sequence motifs (Fig. 1). The residues of these
motifs cluster t ogether t o f orm t he active site. All enzymes o f
the HAD superfamily use the aspartate r esidue of the first
conserved DXXX(T/V) motif as a nucleophilic residue for
catalysis [1]. T he second motif contains a conserved serine o r
threonine residue, and the third motif contains a strictly
conserved lysine residue followed, at some distance, by less
conserved residues and a strictly conserved aspartate.
Mutagenesis studies on these conserved residues show that
all t hree motifs play an important role in the catalytic
process [2–4].
Despite the low overall sequence homology among the
enzymes of the HAD superfamily, all known structures o f
enzymes of this superfamily display a conserved fold [5].
Indeed, 2-haloacid dehalogenase f rom Pseudomonas sp. YL

and Xanthobacter autotrophicus [6,7], phosphonoacetalde-
hyde hydrolase from Bacillus cereus [8], soluble epoxide
hydrolase [9], the Ca
2+
-P-type ATPase [10], b-phospho-
glucomutase from Lactococ cus lacti s [11], phosphoserine
phosphatase (PSP) from Methanococcus j annaschii (MJ
PSP) [12,13] and HPSP [14,15] all have a core a/b domain
resembling the NAD(P)-binding Rossmann fold [5]. This
fold is characterized by a central six-stranded b-sh eet
flanked on both sides by two or three a-helices. The similar
topology and common fold of the central domain, strongly
suggest that the members of t he HAD superfamily evolved
from a primordial, generic domain.
Metals are found in a broad variety of proteins where
they display important functional or structural roles. A
bound Mg
2+
ion is an essential active site component of
numerous metalloproteins including nucleases, kinases and
phosphatases. Such proteins use Mg
2+
for phospho-
substrate binding, catalysis, or both. The HAD superfamily
members, except for 2-haloacid dehalogenases [6,7], utilize
Mg
2+
as a cofactor during catalysis. The effects of various
metal cations on the activity of PSP were described [16], but
key features of their metal binding characteristics remained

undetermined. Maximum activity of the enzyme, measured
by the rate of Pi release from phosphoserine, is obtained
with Mg
2+
. In the absence of added divalent cations, the
activity of PSP is only 9–15% of the maximal a ctivity
observed in the pr esence of Mg
2+
. Of p articular i nterest was
our observation that the replacement of Mg
2+
by Ca
2+
in
an activity test caused complete loss of activity of PSP.
Furthermore Ca
2+
inhibited the activity measured in the
presence of Mg
2+
. Two interesting questions arise from
these observations: is there structural evidence for the fact
that Mg
2+
in the active site cannot be replaced by another
divalent cation without loss of activity, and how does an
enzyme manage to select a specific cation from the
surrounding fluids that contain a broad variety of cations?
AdetailedstudyofthestructureoftheactivesiteofHPSP
with Ca

2+
bound may provide an insight into the biological
Correspondence to A. Rabijns, Laboratory for Analytical Chemistry
and Medicinal Physicochemistry, Faculty of Pharmaceutical Sciences,
K.U. Leuven, E. Van Evenstraat 4, B-3000 Leuven, Belgium.
Fax:+3216323469,Tel.:+3216323421,
E-mail:
Abbreviations: HAD, haloacid dehalogenase; HPSP, human phos-
phoserine phosphatase; Pi, inorganic phosphate; PSP, phosphoserine
phosphatase.
(Received 19 May 2004, revised 1 July 2004, accepted 7 July 2004)
Eur. J. Biochem. 271, 3421–3427 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04277.x
role of metal ions, especially divalent metal ions, in
biological processes.
Materials and methods
Structure determination
The expression, purification and crystallization of HPSP
was carried out using methods described previously [17].
The C a
2+
containing crystal structure elucidated to a
resolution of 1.53 A
˚
has been described elsewhere. The
structure was deposited in the Protein Data Bank (code
1NNL; ) [15]. The final HPSP model
consists of 3203 protein atoms, 390 water molecules, six Cl
–
and three Ca
2+

ions and a summary of the crystallographic
quality indicators for this final model is given in Table 1.
HPSP activity assay
After purification, HPSP was assayed at 3 0 °Cbythe
release of Pi from unlabeled
L
-phosphoserine in an assay
mixture (250 lL) containing 25 m
M
Mes (pH 6.5), 5 m
M
MgCl
2
,1m
M
dithiothreitol, 5 m
ML
-phosphoserine,
0.1 m gÆmL
)1
bovine serum albumin and 1–10 mU HPSP.
Reactions were stopped by the addition of 250 lLof10%
(v/v) trichloroacetic acid and the amount of Pi was
measured using a spectrophotometer [18]. One unit o f
enzyme is the amount that catalyses the conversion of
1 lmol of substrate per minute under these conditions. To
address the effects o f Ca
2+
on the H PSP activity, HPSP was
incubated with different concentrations of Mg

2+
(0.2, 1, 2,
5m
M
) and all these set-ups were assayed in the presence of
increasing concentrations of Ca
2+
(0.00, 0.025, 0.05, 0.10,
0.25 and 1.0 m
M
).
Results and Discussion
Presence of Ca
2+
in the HPSP active site
During the refinement of the HPSP model it b ecame clear
that the electron density peak in the active site could best be
explained by a Ca
2+
ion. The plausible reason for the
presence of the Ca
2+
in the active site, instead of a Mg
2+
ion as in previously reported structures of the PSP family, is
that we used CaCl
2
in the HPSP crystallization condition; at
all times the presence of Mg
2+

was avoided. The two HPSP
molecules in the asymmetric unit (Molecule A and B) were
refined independently of each other. In both molecules, the
atoms surrounding the divalent cation and the metal ion
itself, were in the same range of B factor values (around
15 A
˚
2
); replacing the Ca
2+
ion by another ion (e.g. Mg
2+
)
causes the R factor to increase substantially (0.7%) during
the refinement procedure, as commented by Peeraer et al.
[15]. The presence of a Ca
2+
ion is further confirmed by the
geometry and the metal-donor atom target distances
(Table 2).
Role of the divalent cation in the reaction mechanism
of HPSP
To understand the bio logical role of Mg
2+
in the catalytic
mechanism of PSP, one should k eep in mind that the
hydrolysis of phosphoserine by PSP proceeds through a
stepwise phosphotransfer mechanism, as demonstrated by
Table 1. Data collection, refinement and model statistics for the HPSP
structure a t 1.53 A

˚
resolution. V alues in parentheses i nd icate data in the
highest resolution shell, i.e. 1.56–1.53 A
˚
.
Data collection statistics
Resolution limit (A
˚
) 1.53 (1.56–1.53)
Completeness of all data (%) 99.8 (98.7)
Completeness of the data I > 2r (%) 95.2 (80.7)
Mean I/r 15.0 (3.1)
R
merge
(%) 3.5 (18.7)
R
measure
(%) 3.8 (21.9)
Refinement statistics
Final R
work
(%) 21.6
Final R
free
(%) 23.4
Model statistics
Average atomic B factors (A
˚
2
)

Main chain 24.6
Side chain 27.3
Water molecules 34.4
Ca
2+
atoms 17.6
Cl
–
atoms 23.8
rmsd of the model
Bond lengths (A
˚
) 0.005
Bond angles (°) 1.144
B, bonded main chain (A
˚
2
) 1.194
B, bonded side chain (A
˚
2
) 2.077
Fig. 1. Multiple sequence alignment of the members of the HAD superfamily. The first column indicates the protein and the species it comes from.
PSP, phosphoserine phosphatase; PMM, phosphomannomutase; H AD, haloacid dehalogenase; ATP, ATPase (Human, Homo sapiens ;Meth,
Methanococcus jannaschii;Sacc,Saccharomyces cerevisiae; Coli, Escherichia coli; Pssp, Pseudomonas sp.). Numbers indicate the d istances to the
ends of each prot ein and numbers in parentheses indicate the sizes of the gaps between the aligned segments. The h ighlighted amino acids are
conserved in the HAD superfamily.
3422 Y. Peeraer et al.(Eur. J. Biochem. 271) Ó FEBS 2004
mechanistic studies on MJ PSP [19]. Structural comparison
between HPSP (PDB code 1NNL) and MJ PSP (PDB code

1L7O) structures ( rmsd 1.64 A
˚
for 176 residues super-
imposedandrmsd0.64A
˚
for 16 active site residues
superimposed), reveals that the reaction mechanism o f
HPSP involves subsequent nucleophilic attacks and acid/
base catalysis. The conserved residues Arg65 and Glu29
play an essential role in o rientating the substrate in an
appropriate manner for hydrolysis. The side-chain of Glu29
interacts with the amino group of phosphoserine, while the
side-chain of Arg65 forms a hydrogen bond with the
carboxyl group of the s ubstrate. When the substrate is
positioned correctly, the enzyme closes and Asp20 performs
a nucleophilic attack on the scissile phosphate. The
substrate phosphoserine is then cleaved, resulting in the
departure of the leaving group serine and the formation of a
covalent phosphoaspartyl (Asp20) intermediate (Fig. 2 ).
Asp22 serves as a general acid (Fig. 2, Enz-H) donating a
proton to serine and thereby facilitating the expulsion of the
leaving group. A water molecule takes the position in the
just-vacated leaving group site. The Asp22 carboxylate
anion (Fig. 2, Enz-B) that was formed during t he protona-
tion of the leaving serine group, can now serve as a base
catalyst in the dephosphorylation of the phosphoenzyme
intermediate. Asp22 extracts a p roton from the water
molecule in the active site, thereby activating the water
molecule to perform a nucleophilic attack on the phospho-
aspartyl intermediate. Opening of the enzyme and dissoci-

ation of the inorganic phosphate completes the catalytic
cycle.
The Mg
2+
ion in the active site is essential for HPSP to
perform the hydrolysis of phosphoserine. First of all Mg
2+
plays a catalytic role in the reaction mechanism. The Mg
2+
ion coordinates both an oxygen atom of the phosphate
moiety of the s ubstrate a nd an oxygen a tom o f the attackin g
Asp20 residue. In this way the Asp20 residue is stabilized in
an optimal position t o p erform an attack on the phosphorus
atom of phosphoserine. In addition, the positive charge of
the divalent cation is essential to facilitate the nucleophilic
attack of Asp20 by extracting negative charge from the
phosphate group. The fact that haloacid dehalogenases do
not need a divalent cation for activity, while the phospho-
transferases of the same HAD superfamily do, supports the
idea that a divalent c ation in HPSP i s needed to shield the
negative charges of the phosphate group while the attacking
nucleophile Asp20 is approaching. Of interest is that in
haloacid dehalogenase, the corresponding attacking Asp
residue approaches an electropositive carbon centre of the
substrate and thus a cation is not required to promote the
nucleophilic attack.
Besides its catalytic role, the divalent cation in the HPSP
active site also plays a purely structural role. In the H PSP
active site, three Asp residues (20, 2 2 and 179) are in close
proximity to each other and form a carboxylate cluster,

thereby generating an excess of negative charge in the
binding pocket. The positive charge of the divalent cation is
therefore necessary to stabilize the overall architecture of
this carboxylate cluster by diminishing the electrostatic
repulsion between the negative charges of the Asp side-
chains. The stabilizing, structural role of Mg
2+
is further
illustrated b y the fact that Asp179, which belongs to
sequence motif III and which coordinates the divalent
cation in the active site, is conserved in all the HAD
superfamily members w ith t he exception of the enzymes that
are Mg
2+
-independent for their activity. Indeed, in the
haloacid dehalogenases, the corresponding residue is a Ser
which is not essential for catalytic activity [7]. This
observation suggests that Asp179 is essential for binding
of the divalent cation in the active site. Furthermore,
mutagenesis studies on HPSP showed that mutation of
Asp179 to an Asn or Glu results in a 10-fold decrease in the
affinity for Mg
2+
[4]. The same functions for the Mg
2+
ion
are observed in other Mg
2+
-dependent members of the
HAD superfamily like phosphonoacetaldehyde hydrolase,

b-pho sphoglucomutase and P-type ATPases [8,11,20].
Mg
2+
substituted by a Ca
2+
: implications for the reaction
mechanism
Neuhaus & Byrne [16] reported that HPSP activity depends
on the presence of Mg
2+
. We confirmed this requirement
and we d etermined t hat t he K
a
for Mg
2+
in the p resence o f a
saturating concentration of substrate was 0.2 m
M
(not
shown). From Fig. 3 it can be seen that Ca
2+
inhibited the
enzyme activity, and the lower the Mg
2+
concentration the
more apparent this effect was. Indeed, a 50% inhibition was
observed a t 0.01, 0.025, 0.05 and 0.2 m
M
Ca
2+

in the
presence of 0.2, 1, 2 and 5 m
M
Mg
2+
.
Several experiments to also obtain a Mg
2+
-containing
HPSP structure, i.e. soaking and cocrystallization experi-
ments, failed. Therefore, to elucidate the inhibitory effect
exerted by Ca
2+
on the activity of PSP, w e compared the
active site of HPSP, which contains a Ca
2+
ion, with the M J
PSP active site, con taining Mg
2+
(PDB codes 1F5S, 1L7P
Table 2. Distances between the Ca
2+
ion and the neighbouring atoms in the active site for molecules A and B of the asymmetric unit of the HPSP
structure. Th e typical metal-donor atom target distances for Ca
2+
and Mg
2+
are also given [24]. This s hows that the observed distances in the HPSP
structure correspond to typical Ca
2+

distances.
Ligating
atom
Ca
2+
(Mol A) (A
˚
)
Ca
2+
(Mol B) (A
˚
)
Target distance
(A
˚
) for Ca
2+
ion
Target distance
(A
˚
) for Mg
2+
ion
Asp20 OD1 2.37 2.38 2.36 2.26
Asp22 O 2.31 2.29 2.36 2.26
Asp179 OD2 2.30 2.33 2.36 2.26
H
2

O 2.33 2.30 2.39 2.07
H
2
O 2.44 2.49 2.39 2.07
H
2
O 2.37 2.41 2.39 2.07
Ó FEBS 2004 Inhibition of human phosphoserine phosphatase (Eur. J. Biochem. 271) 3423
and 1J97). The Mg
2+
ion in the MJ PSP active site displays
almost perfect octahedral coordination geometry with six
ligands. Four ligands are in a plane with O–Mg
2+
–O angles
of nearly 90°, while the two other ligands are above and
below this plane, r espectively. The c oordination of the Ca
2+
ion in the active site of HPSP is distorted from octahedral
geometry as shown in Fig. 4. In open co nformation, three
water molecules and three O atoms (OD1 of Asp20, the
main-chain carbonyl group of Asp22 and OD2 of Asp179)
occupy six of the coordination sites of the Ca
2+
, similar to
the Mg
2+
ion in the MJ PSP active site. Nevertheless, it can
be seen that one water molecule (Fig. 4B, Wat1) i s forced
out of the plane. This distortion of the octahedral geometry

is due to the fact that the Ca
2+
ion prefers seven ligands
instead of six as Mg
2+
does. Because the coordination of
spherical metal ions is optimized by maximum packing of
ligand atoms, the preferred coordination number is p rimar-
ily a function of the size of the ion [21]. The effective ionic
radius of a Mg
2+
ion (0.72 A
˚
) is considerably smaller than
that of a Ca
2+
ion (1.06 A
˚
) [22]. The smaller size of Mg
2+
determines its preference for a coordination number of six.
In contrast, the effective ionic radius of Ca
2+
is s uch that
seven or e ight coordinating ligands can be comfortably
accommodated [23]. As a result the Ca
2+
ionintheHPSP
active site accepts both side-chain oxygen atoms of Asp20
as a ligand, while a Mg

2+
ion ligates only one oxygen atom
of this Asp residue.
Besides the differences in geometry between Ca
2+
and
Mg
2+
in the active site, the metal–ligand distances are
also quite different. Comparison of the active sites of
HPSP and MJ PSP shows t hat replacement of a Mg
2+
by
aCa
2+
ion results in an increase in all metal–ligand
Fig. 2. General scheme of the reaction cycle of PSP [22]. Open conformation of PSP (A).
L
-Phosphoserine binds to the active site presenting the
phosphate group to Asp20 (B). Transition state with nucleophylic attack of Asp20 (C). Covalent phospho aspartyl enzyme i ntermediat e (D).
Transition state with a nucleophylic attack of a water molecule cau sing the de phosphorylation of Asp20 (E). Phosphate noncovalently bo und in the
active site (F). Enz-H indicates the general acid Asp22, which after the protonation of the leaving serine group serves as a base catalyst Enz-B.
Fig. 3. Effect of C a
2+
on HPSP activity. Th e eff ect o f Ca
2+
on HPSP
activity was assayed i n the pres ence of 0.2 (j), 1 ( m), 2 ( .)or5(r)m
M
Mg

2+
. HPSP activity was assayed as in Materials and methods.
3424 Y. Peeraer et al.(Eur. J. Biochem. 271) Ó FEBS 2004
distances, with average distances of 2.1 A
˚
for Mg
2+
and
2.4 A
˚
for Ca
2+
. The observed distances match to a large
extent the ideal distances for Ca
2+
-donor atom combina-
tions and similar distances are observed i n various
metalloproteins [24]. Combination of the dissimilar geo-
metry and changed metal–ligand distances drastically
affects the reaction mechanism of HPSP when the
Mg
2+
ion is substituted by a Ca
2+
ion (Fig. 5). Upon
substrate binding, on e of the three water m olecules
coordinating the divalent ion is replaced by an oxygen
of the phosphate moiety of phosphoserine. The fact that
Asp20 in HPSP acts as a bidentate ligand in the sevenfold
coordination of Ca

2+
(OD1 and OD2 to Ca
2+
distances
of 2.38 and 2.77 A
˚
, respectively), while the corresponding
Asp11 in MJ PSP is a monodentate ligand in the sixfold
Mg
2+
coordination, hampers th e nucleophilic attack of
OD1 on the substrate. The corresponding OD1 of Asp11
in the Mg
2+
bound MJ PSP is 3.33 A
˚
away from the
cation and therefore it is free to perf orm an attack on the
phosphorus atom of the substrate. The distance between
Fig. 4. Detailed o verview of the Ca
2+
+
ion i n the active site of HPSP. (A) The residues are represented in ball and stick form with oxyge n, carbon a nd
nitrogen atoms coloured red, light-blue and dark-blue, respectively. The Ca
2+
ion is shown in green. Thre e of t he Ca
2+
ligands ar e water molecules,
shown as red balls. The dashed lines represent hydrogen bonds and metal–ligand interactions. Asp20, Asp22 and Asp179 directly coordinate the
Ca

2+
ion. Asp179 and Gly180 interact with Asp183, thereby stabilizing the loop on which they are located. (B) The coordination of the Ca
2+
is
distorted from ideal oct ahedral geometry with six l igands because it forms an extra interaction with one of the oxygen atoms of Asp20. This extra
interaction between Ca
2+
and Asp20, shown in green, does not occur with a Mg
2+
ion in the active site.
Fig. 5. Active s i te of M J PSP w ith a M g
2+
+
and pho sphoserine in the active site (PDB codes 1F5S and 1L7P) (A) and HPSP (PDB c ode 1N NL) with a
Ca
2+
ion bound and the modelled substrate in the active site ( B). For clarity only four ligands are shown, i.e . a ligating water mol ecule and Asp13/22
(HPSP/MJ PSP) are omitted i n this fi gure. In c ontrast to a Mg
2+
ion, the Ca
2+
ion i n HPSP liga tes both oxy gen atoms o f Asp20 there by preventing
it to perform a nucleophilic attack on the phosphorus atom of the substrate. In addition, a C a
2+
ion displays longer metal–ligand d istances than a
Mg
2+
ion. As a c onsequ ence the partial p o sitive charge o n t he ph osphorus ato m o f p hosphoserine i s sm aller if a Ca
2+
takes position i n the active

site. In this manner, a Ca
2+
will further hamper the nucleophilic attack of the catalytic Asp residue on the substrate.
Ó FEBS 2004 Inhibition of human phosphoserine phosphatase (Eur. J. Biochem. 271) 3425
the OD1 of the attacking Asp r esidue and the phos-
phorus atom of the substrate is increased from 2.96 A
˚
with a Mg
2+
ionto3.36A
˚
with a Ca
2+
,further
hampering the nucleophilic attack on the substrate. In
MJ PSP the distance between oxygen O2 of the phosphate
part of
L
-phosphoserine and the Mg
2+
ion is 2.41 A
˚
[19].
Replacing the Mg
2+
ion by a Ca
2+
ion results in an
increase of this distance to 3.20 A
˚

. This will undoubtedly
result in a smaller attraction of negative charge from the
phosphate moiety of the substrate, thereby suppressing the
nucleophilic attack of Asp20 on the phosphorus atom.
HPSP selectivity for Mg
2+
For the Mg
2+
binding site of the related CheY enzyme
[25,26], it was proposed that the c arboxylate c luster in the
active site provides charge specificity to the Mg
2+
binding
site by excluding monovalent cations like Na
+
and K
+
,
because they do not possess sufficient positive charge to
stabilize the highly negative carboxylate cluster [27]. Ana-
logously, HPSP can exploit the negative charge of the
carboxylate cluster, composed of Asp20, Asp22 a nd
Asp179, to provide the necessary charge specificity by
excluding monovalent cations.
On the other hand, it seems that t he Mg
2+
binding site
in HPSP is weakly protected against the binding of other
divalent cations like Ca
2+

,asCa
2+
displays inhibiting
properties even in the presence of Mg
2+
([16] and this
paper). The weak size-selectivity of HPSP can originate
from the f act that in HPSP in open conformation three of
the ligands to the divalent cation are water m olecules [15].
An interesting feature of this coordination structure is that
one hemisphere of the bound ion is coordinated by three
protein oxygens, while the other hemisphere is coordina-
ted by three solvent molecules (Fig. 4). These water
molecules can easily accommodate changing metal–ligand
distances if Mg
2+
is replaced by a larger divalent cation
such as Ca
2+
. In addition, the larger Ca
2+
ion can
employ Asp20 as a bidendate ligand in o rder to complete
its preferred sevenfold coordination geometry. Thus, the
difference in ionic radii of Ca
2+
and Mg
2+
is not a
sufficient criterion for HPSP to select Mg

2+
,asthe
binding cavity of the enzyme is flexible and able to adjust
easily to different ionic radii and changing coordination
geometry.
In view of the facts outlined above i t becomes clear that
the metal-binding pocket of HPSP is charge-selective in
order to discriminate between mono- and divalent c ations,
but not size-selective enough to single out particular divalent
cations as Mg
2+
and Ca
2+
. Nevertheless, in living cells
HPSP uses Mg
2+
as a cofactor and not the larger Ca
2+
.
Thelatterseemslogical,asMg
2+
is the most abundant
divalent cation in eukaryotic cells, with concentrations of
free Mg
2+
ranging from 0.1 to 1.0 m
M
, while the Ca
2+
concentration is 10

4
-fold lower in resting eukaryotic cells
[28]. Thus, HPSP has chosen Mg
2+
as a cofactor during
evolution based mainly on its n atural abundance in living
cells. In this scenario it is not the protein metal-binding
pocket architecture itself but the cell homeostasis that
controls the process of metal binding by regulating the
appropriate concentrations of Mg
2+
and other cations in
various biological compartments.
Conclusions
The HPSP reaction mechanism involves nucleophilic attack
of Asp20 on the substrate with a cid/base catalysis mediated
by Asp22. The Mg
2+
ion in the active site is essential for
normal enzymatic activity, i.e. the Mg
2+
ion promotes the
nucleophilic attack of Asp20 by withdrawing negative
charge from the phosphorus atom of the substrate. In
addition, the divalent cation is essential f or the correct
orientation of the attacking Asp20 residue towards the
substrate. A Ca
2+
ion h owever, employs Asp20 as a
bidentate ligand, thereby inhibiting the nucleophilic attack

of this catalytic residue. Furthermore, it seems that the
Mg
2+
binding site in HPSP is weakly protected against the
binding of other divalent cations, as Ca
2+
displays inhib-
iting properties even in t he presence of M g
2+
. Therefore it is
probable that HPSP h as chosen Mg
2+
as a cofactor during
evolution based mainly on the natural abundance of Mg
2+
in living cells.
Acknowledgements
A.R. is a Postdoctoral Research Fellow of the Fund for Scientific
Research-Flanders (Belgium) and J F.C. w as Charge
´
de R echerches of
the Belgian FNRS. Work in the lab of E.V.S. is supported by the
Interuniversity A ttraction Poles Program-Belgian Science Polic y and b y
the FRSM. We thank the beam line scientists at DESY for technical
support and the European Union for support of the work at EMBL
Hamburg through the Access to Research Infra structure Action of the
improving human potential programme, contact no. HPRI-CT-1999-
00017.
References
1. Collet, J.F., Stroobant, V., Pirard, M., Delpierre, G. & Van

Schaftingen, E. (1998) A new class of phosphotransferases phos-
phorylated on an aspartate residue in an amino-terminal
DXDX(T/V) motif. J. Biol. Chem. 273, 14107–14112.
2.MacLennan,D.H.,Clarke,D.M.,Loo,T.W.&Skerjanc,I.S.
(1992) Site-directed m utagenesis of the Ca
2+
ATPase of sarco-
plasmic reticulum. Acta Physiol. Scand. Suppl. 607, 141–150.
3. Lingrel, J.B. & Kuntzweiler, T. (1994) Na
+
,K
+
-ATPase. J. Biol.
Chem. 269, 19659–19662.
4.Collet,J.F.,Stroobant,V.&VanSchaftingen,E.(1999)
Mechanistic studies of phosphoserine phosphatase, an enzyme
related to P-type ATPases. J. Biol. Chem. 274, 33985–33990.
5. Rossmann, M.G., M oras, D . & Olsen, K.W. (1974) Chemical and
biological evolution of a nucleotide-binding protein. Nature 250,
194–199.
6. Hisano, T., Hata, Y., Fujii, T., Liu, J.Q., Kurihara, T., Esaki, N.
& Soda, K. (1996) Crystal structure of L-2-haloacid dehalogenase
from Pseudomonas sp. YL. J. Biol. Chem. 271, 20322–20330.
7. Ridder, I.S. & Dijkstra, B.W. (1999) Identification of the Mg
2+
-
binding site in the P-type ATPase and phosphatase members of
the HAD (haloacid dehalogenase) superfamily by structural
similarity to the response regulator CheY. Biochem. J. 339, 223–
226.

8. Morais, M.C., Zhang, W., Baker, A.S., Zhang, G., Dunaway-
Mariano, D. & A llen, K.N. (2000) The crystal structure of Bacillus
cereus phosphonoacetald ehyd e hydrolase: insight into catalysis of
phosphorus bond cleavage and catalytic diversification within the
HAD enzyme superfamily. Biochemistry 39, 10385–10396.
9. Argiriadi, M.A., Morisseau, C., Hammock, B.D. & Christianson,
D.W. (1999) Detoxification of environmental mutagens and
3426 Y. Peeraer et al.(Eur. J. Biochem. 271) Ó FEBS 2004
carcinogens: structure, mechanism and evolution of liver epoxide
hydrolase. Proc. Natl Acad. Sci. USA 96, 10637–10642.
10. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000)
Crystal structure of the calcium pump of sarcoplasmic reticulum
at 2.6 A
˚
resolution. Nature 405, 647–655.
11. Lahiri, S.D., Zhang, G., Dunaway-Mariano, D. & Allen, K.N.
(2002) Caught in the act: the structure of phosphorylated
b-pho sphoglu comutase from Lactococcus lactis. Biochemistry 41 ,
8351–8359.
12. Cho, H., Wang, W., Kim, R., Yokota, H., Damo, S., Kim, S.H.,
Wemmer, D., Kustu, S. & Yan, D. (2001) BeF
À
3
acts as a
phosphate analog in proteins phosphorylated on aspartate:
structure of a BeF
À
3
complex with phosphoserine phosphatase.
Proc.NatlAcad.Sci.USA98, 8525–8530.

13. Wang, W., Kim, R., Jancarik, J., Yokota, H. & Kim, S.H. (2001)
Crystal structure of phosphoserine phosphatase from Methano-
coccus jannaschii, a hyperthermophile, at 1.8 A
˚
resolution. Struc-
ture 9, 65–71.
14. Kim, H.Y., Heo, Y .S., Kim, J.H., Park, M.H., M oon, J., Kwan,
D., Yoon , J., Shin, D., Jeong, E., Park, S.Y., Lee, T.G., Jeon,
Y.H., Ro, S., Cho, J.M. & Hwang, K.Y. (2002) Molecular basis
for the local conformatio nal rearrangem ent of hum an pho spho-
serine phosphatase. J. Biol. Chem. 227, 46651–46658.
15. Peeraer,Y.,Rabijns,A.,Verboven,C.,Collet,J.F.,VanSchaf-
tingen, E . & De Ranter, C. (2003) High resolution structure of
human p hosp hoserine phosphatase in open conformation. Acta
Cryst. D59, 971–977.
16. Neuhaus, F.C. & Byrne, W.L. (1959) Metabolism o f phospho-
serine. II. Purification and properties of O-phosphoserine phos-
phatase. J. Biol. Chem. 234, 113–121.
17. Peeraer,Y.,Rabijns,A.,Verboven,C.,Collet,J.F.,VanSchaf-
tingen, E. & De Ranter, C. (2002) P urification, crystallization a nd
preliminary X-ray an alysis of human ph osphoserine p hosp hatase.
Acta Cryst. D58, 133–134.
18. Fiske, C.H. & Subbarow, Y.P. (1925) The colorimetric determi-
nation of phosphorus. J. Biol. Chem. 66, 375–400.
19. Wang,W.,Cho,H.,Kim,R.,Jancarik,J.,Yokota,H.,Nguyen,
H.H., Grigoriev, I.V., Wemmer, D.E. & Kim, S.H. (2002) Struc-
tural characterization of the reaction pathway in phosphoserine
phosphatase: crystallographic ‘‘snapshots’’ of intermediate states.
J. Mol. Biol. 319, 421–431.
20. Stokes, D.L. & Green, N.M. (2003) Structure and function of the

calcium pump. Annu. Rev. Biophys. Biomol. Struct. 32, 445–468.
21. Yang, W., Lee, H.W., Hellinga, H. & Yang, J.J. (2002) Structural
analysis, identification and design of calcium-bind ing sites in
proteins. Proteins: Stuct. Func. Genet. 47, 344–356.
22. Shannon, R.D. (1976) Revised effective ionic radii and systematic
studies of interatomic d istances in halides and chalcogenides. Acta
Cryst. A36, 751–767.
23. Falke, J.J., Drake, S.K., Hazard, A.L. & Peersen, O.B. (1994)
Molecular tuning of ion binding to calcium signaling proteins.
Q Rev. Biophys. 27, 219–290.
24. Harding, M.M. (2001) Geometry of metal-ligand interactions in
proteins. Acta Cryst. D57, 401–411.
25. Bellsollel, L., Prieto, J., Serr ano, L. & Coll, M. (1994) M agnesium
binding to the bacterial chemotaxis protein CheY results in large
conformational changes involving its functional surface. J. M ol.
Biol. 238, 489–495.
26. Ridder, I.S., Rozeboom, H.J., Kalk, K.H., Janssen, D.B. &
Dijkstra, B .W. (1997) Three-dim ensi onal structure o f
L
-2-haloacid
dehalogenase from Xanthobacter autotrophicum GJ10 complexed
with the substrate analogue formate. J. Biol. Chem. 272, 33015–
33022.
27. Needham, J.V., C hen, T.Y. & Falk e, J.J. (1993) Novel ion speci-
ficity of a carboxylate cluster Mg(II) binding site: strong charge
selectivity and weak size selectivity. Biochemistry 32, 3363–3367.
28. Romani, A. & Scarpa, A. (1992) Regulation of cell magnesium.
Arch. Biochem. Biophys. 298, 1–12.
Ó FEBS 2004 Inhibition of human phosphoserine phosphatase (Eur. J. Biochem. 271) 3427