Active and regulatory sites of cytosolic 5¢-nucleotidase
Rossana Pesi
1
, Simone Allegrini
2
, Maria Giovanna Careddu
1,2
, Daniela Nicole Filoni
1
,
Marcella Camici
1
and Maria Grazia Tozzi
1
1 Dipartimento di Biologia, Unita
`
di Biochimica, Universita
`
di Pisa, Pisa, Italy
2 Dipartimento di Scienze del Farmaco, Universita
`
di Sassari, Sassari, Italy
Introduction
Cytosolic 5’-nucleotidase (cN-II) is a ubiquitous enzyme
that catalyses either the hydrolysis or the transfer of
phosphate esterified in the 5¢ position of 6-hydroxypu-
rine monophosphate nucleosides [1]. The transfer of
phosphate can lead to phosphorylation of inosine,
guanosine and a number of their analogues [2]. There-
fore, in addition to being involved in regulation of
purine intracellular pool, the enzyme is also responsible

4
A and ADP interact
with the same site, but the sites for ATP and BPG remain uncertain. The
structural model indicates that cN-II is a homotetrameric protein that
results from interaction through a specific interface B of two identical
dimers that have arisen from interaction of two identical subunits through
interface A. Point mutations in the two interfaces and gel-filtration experi-
ments indicated that the dimer is the smallest active oligomerization state.
Finally, gel-filtration and light-scattering experiments demonstrated that
the native enzyme exists as a tetramer, and no further oligomerization is
required for enzyme activation.
Structured digital abstract
l
MINT-8011572: cN-II (uniprotkb:O46411) and cN-II (uniprotkb:O46411) bind (MI:0407)by
dynamic light scattering (
MI:0038)
l
MINT-8011493, MINT-8011481: cN-II (uniprotkb:O46411) and cN-II (uniprotkb:O46411)
bind (
MI:0407)bymolecular sieving (MI:0071)
Abbreviations
cN-II, cytosolic 5¢-nucleotidase; cN-III, cytosolic 5¢-nucleotidase III; cN-IA, cytosolic 5¢-nucleotidase IA; cN-IB, cytosolic 5¢-nucleotidase IB;
cdN, cytosolic 5¢(3¢)-deoxyribonucleotidase; mdN, mitochondrial 5¢(3¢)-deoxyribonucleotidase.
FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4863
for pro-drug activation and inactivation [3,4]. It has
been demonstrated that the catalytic mechanism of
cN-II requires formation of a covalent phospho-inter-
mediate on an aspartate residue located in a conserved
motif (motif I) [5]. This motif, together with three
other conserved motifs, is shared among the members

Free inorganic phosphate, on the other hand, acts as
an allosteric inhibitor, causing a 20-fold increase in K
m
for the substrate IMP with little effect on V
max
. Inter-
estingly, ATP partially counteracts the effect of phos-
phate, by increasing V
max
; however, it is unable to
reverse the increase in K
m
[9]. cN-II has been described
as a homotetramer with the ability to change its oligo-
merization state in response to the presence of activa-
tors or inhibitors. It has been suggested that the
change in the oligomerization state is accompanied by
a change in specific activity [10]. However, this simple
model does not explain the kinetic evidence described
above.
Despite its cytosolic location, cN-II has particularly
poor solubility. This is why it has been difficult to
obtain the crystal structure of the whole protein. A
truncated form of cN-II lacking the last 25 amino
acids is significantly more soluble than the wild-type
enzyme, and was recently crystallized [11]. The crystal-
lographic model, constructed by ordering 487 residues
(1-400 and 417-488) out of 561, indicates a homotetra-
meric protein resulting from interaction through a
specific interface (interface B) of two identical dimers

Active site and phosphotransferase reaction
Figure 1 shows the aligned sequences of motif I for the
six intracellular 5¢-nucleotidases. Of the six enzymes,
only cN-II and cN-III possess a Thr instead of a Val
(position 56 of cN-II, boxed in Fig. 1) [11]. These are
the only two enzymes for which phosphotransferase
activity has been unquestionably ascertained, and
Wallde
´
n et al. [11] suggested that the presence of T56
Fig. 1. Aligned conserved motif I of the six known intracellular
human 5¢-nucleotidases: cN-II, cN-III, cN-IA, cN-IB, cytosolic 5¢(3¢)-
deoxyribonucleotidase (cdN) and mitochondrial 5¢(3¢)-deoxyribonu-
cleotidase (mdN). Residues 55 and 56 of cN-II are indicated in bold.
Regulation of cytosolic 5¢-nucleotidase R. Pesi et al.
4864 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS
might be important for this activity. However, we
noted that there is another variable residue near T56.
In position 55 of cN-II, a Tyr is present that is substi-
tuted by Met in cN-III and by Ala or Gly in the other
enzymes. Therefore, the two phosphotransferases have
a bulkier amino acid in this position compared to the
other four enzymes, which have amino acids with very
small side chains. We decided to construct two
mutants of motif I at positions 55 (Y55G) and 56
(T56V) in order to ascertain whether one of them is
responsible for the phosphotransferase activity. Deter-
mination of kinetic parameters showed that substitu-
tion of Tyr55 by a smaller residue causes a dramatic
increase in the ratio of nucleotidase to phosphotrans-

Phe127 and His428, as adenosine is presumably
stacked between these two residues, and Met436,
which forms a hydrogen bond with the purine ring
through its carbonylic group [11] (Fig. 3). We substi-
tuted the first two residues by a negatively charged res-
idue to discourage stacking, and replaced Met436 with
a bulkier amino acid (Trp).
Putative effector site 1
None of the mutations produced had a significant
effect on K
m
for the two principal substrates (Table 2).
Table 1. Effect of point mutations on various kinetic parameters of recombinant bovine cN-II. Nucleotidase and phosphotransferase activi-
ties were measured as described in Experimental procedures. Values are means ± SD of at least three independent assays. k
cat
refers to
nucleotidase activity and is measured at saturating concentrations of IMP and sub-saturating concentrations of inosine. The K
50
for ATP was
measured as phosphotransferase activity, while for Mg
2+
, it was measured as nucleotidase activity.
cN-II
Nucleotidase ⁄
phosphotransferase
K
m
(inosine)
(m
M)

P
i
Ino H
2
O
E + IMP
E + IMP E-P
E + P
i
Ino
Fig. 2. Rate of inosine, IMP and P
i
production catalysed by wild-
type cN-II (black bars) or mutant Y55G (white bars) in the presence
of 2 m
M IMP and 1.4 mM inosine (Ino) as substrates. The assays
were performed as described in Experimental procedures. For wild-
type and Y55G, 100% activity corresponds to 32 and 18 UÆmL
)1
,
respectively. The upper scheme indicates the catalytic mechanism
of cN-II. E, enzyme; P, phosphate. The products measured are
boxed.
R. Pesi et al. Regulation of cytosolic 5¢-nucleotidase
FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS 4865
Mutant R144E showed an altered affinity for all the
activators tested, N154D was normally activated by
ATP and BPG but activation by ADP and Ap
4
A was

In the model proposed on the basis of the crystal struc-
ture, interface A is involved in formation of a dimeric
structure (Fig. 3). Fifty-three amino acids contribute to
interaction of the two monomers by forming both
hydrogen bonds and salt bridges. Some of these residues
are near to effector site 1. We designed our mutants in
Fig. 3. Model of the homotetrameric quaternary structure of cN-II
showing interfaces A and B and the Mg
2+
site. The inset shows
the tertiary structure of each subunit. Effector sites 1 and 2 and
the active site are shown.
Table 2. Effect of point mutations on various kinetic parameters of recombinant bovine cN-II. Nucleotidase and phosphotransferase activi-
ties were measured as described in Experimental procedures. Values are means ± SD of at least three independent assays. K
50
values for
P
i
, ATP, ADP and Ap
4
A were measured as phosphotransferase activity, while those for BPG and Mg
2+
were measured as nucleotidase activ-
ity. The extent of activation, when present, was between 5- and 10-fold. (1) Mutation in putative effector site 1; (2) mutation in putative
effector site 2. NA, no activation.
cN-II
K
m
(IMP)
(m

K
50
(ADP)
(m
M)
K
50
(Ap
4
A)
(m
M)
Wild-type 0.10 ± 0.02 1.0 ± 0.2 2.0 ± 0.5 2.0 ± 0.3 0.3 ± 0.06 1.0 ± 0.3 2.2 ± 0.5 0.1 ± 0.05
R144E (1) 0.10 ± 0.05 1.0 ± 0.2 2.0 ± 1.0 6.5 ± 1.0 5.0 ± 1.50 30.0 ± 5.0 33.0 ± 6.0 1.6 ± 1.00
N154D (1) 0.1 ± 0.04 2.5 ± 0.5 0.3 ± 0.3 4.0 ± 1.0 0.3 ± 0.07 0.5 ± 0.2 NA NA
I152D (1) 0.2 ± 0.05 0.7 ± 0.2 0.9 ± 0.8 3.5 ± 1.2 5.0 ± 2.00 20.0 ± 4.0 NA NA
F127E (2) 0.1 ± 0.03 1.0 ± 0.3 0.9 ± 0.7 1.5 ± 0.5 0.7 ± 0.10 1.5 ± 1.0 2.5 ± 0.7 0.2 ± 0.03
M436W (2) 0.1 ± 0.02 0.9 ± 0.3 1.5 ± 1.0 1.5 ± 0.5 0.5 ± 0.06 1.5 ± 1.0 1.5 ± 1.0 1.0 ± 0.50
H428D (2) 0.3 ± 0.05 0.6 ± 0.5 1.5 ± 1.0 0.4 ± 0.3 NA NA NA NA
Regulation of cytosolic 5¢-nucleotidase R. Pesi et al.
4866 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS
an attempt to interfere with monomer aggregation via
interface A by introducing or deleting charged residues
or altering molecular hindrance. One of the mutated
amino acids (Tyr115) is located very close to Arg144,
which is part of effector site 1 (Fig. S1). The mutant
F36R was inactive, while Y115A behaved normally and
D396A was only activated by BPG (Table 3).
Interface B
The structure described by Wallde

tetramer irrespectively of the presence of activator or
inhibitor. We also performed light-scattering measure-
ments of the enzyme alone or in the presence of its effec-
tors. There was no change in molecular mass in the
presence of enzyme activators or inhibitors (Table 4).
Discussion
Active site
The soluble 5¢-nucleotidase family shares four con-
served motifs with the HAD superfamily that are
involved in the reaction mechanism [5]. Resolution of
the crystal structure of some family members indicated
that the active site contains all the conserved motifs,
whose role in catalysis was determined through a
mechanistic approach [5,11,13]. Other than these con-
served motifs, cN-IA, cN-IB, cN-II, cN-III and the
cytosolic and mitochondrial 5¢(3¢)-deoxyribonucleotid-
ases differ considerably in their primary structure.
Despite this poor similarity, there is much evidence to
indicate that all members of soluble 5¢-nucleotidase
family share the same reaction mechanism, proceeding
through a covalent phospho-enzyme intermediate
[13,14]. Phospho-cN-II has been isolated, and the
phosphate has been found to be localized to a con-
served aspartate residue (D52) in the first of the four
conserved motifs [13]. Formation of a phospho-inter-
mediate suggests the possibility that the enzyme cataly-
ses transfer of phosphate to a suitable acceptor [15].
A number of HAD superfamily members are able to
catalyse a phosphotransferase reaction, including at
least two soluble nucleotidases (cN-II and cN-III). The

M)
K
m
(inosine)
(m
M)
K
50
(Mg
2+
)
(m
M)
K
50
(P
i
)
(m
M)
K
50
(BPG)
(m
M)
K
50
(ATP)
(m
M)

A and polyphosphates. Con-
versely, orthophosphate has an inhibitory effect.
Kinetic studies have indicated that ATP (and presum-
ably other phosphorylated compounds) causes stabil-
ization of an enzyme form with a high k
cat
, without
substantial alteration of the K
m
for the substrates,
while orthophosphate stabilizes a form at high K
m
with
no effect on k
cat
. If ATP and phosphate are present at
the same time, an enzyme form with high K
m
and high
k
cat
is observed [9]. Therefore, depending on the effec-
tor, cN-II may be present as one of two structures
.
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Fig. 5. FPLC profiles of wild-type cN-II alone (A), or in the presence
of 5 m
M ATP and 10 mM MgCl
2
(B), or in the presence of 5 mM P
i
and 10 mM MgCl
2
(C). The column, prepared and eluted as
described in Experimental procedures, was loaded with approxi-
mately 150 lg of purified protein, and the activity was measured as
the rate of IMP production in the presence of inosine (phospho-
transferase activity) as described in Experimental procedures.
Arrows indicate the molecular mass of the marker proteins thyro-
globulin (670 kDa), apoferritin (443 kDa), b-amylase (210 kDa) and

72
Retention time (min)
Abs
254 nm
(Arbitrary units) (–)
WT
G319D
K311A

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molecular mass of the marker proteins thyroglobulin (670 kDa),
apoferritin (443 kDa), b-amylase (210 kDa) and alcohol dehydroge-
nase (150 kDa).
Regulation of cytosolic 5¢-nucleotidase R. Pesi et al.
4868 FEBS Journal 277 (2010) 4863–4872 ª 2010 The Authors Journal compilation ª 2010 FEBS
with high and low K
m
. In addition, a low and high k
cat
may be associated with each structure. It has also been
suggested, on the basis of kinetic characterization, that
the enzyme has at least three effector sites, one for
ATP, one for ADP and one for BPG [5,12]. On the
basis of the results obtained from crystallization of a
truncated form of cN-II [11], we constructed point
mutants for a number of amino acid residues located
in putative effector sites 1 and 2 and involved in bind-
ing of adenylic nucleotides and BPG.
Our results partially confirm the molecular model-
ling, suggesting that effector site 1 is the binding site
for Ap
4
A and that ADP binds this site as well. Ap
4
A
binds between two subunits with one adenosine moiety
in each subunit [11], but ADP may possibly fill the
whole site in each subunit and attain the same result.
Mutant N154D shows a decrease in the value of K
50

may not be indicative of the location of the nucleoside
triphosphate effector site.
CN-II subunit oligomerization
CN-II has been purified from various sources and has
always been described as a homotetramer [16,17]. The
crystal structure suggests that the tetramer arises from
interaction of two dimers through interface B. Muta-
tions in interface A, through which two monomers
interact, and which is very close to the effector site 1,
either resulted in a completely inactive enzyme or
strongly interfered with activation by ADP and Ap
4
A.
This indirectly confirms that effector site 1 is specific
for these compounds. Mutation of amino acid residues
located in interface B generated proteins for which an
active dimeric form was detected in addition to the tet-
ramer. However, stabilization of the dimeric form had
no effect on the catalytic capacity of the enzyme. Our
results show that the monomer is probably inactive,
and the dimer is the smallest active cN-II quaternary
structure. FPLC analysis of purified recombinant
enzymes shows a heavier protein (720 kDa) in addition
to the proteins at the expected molecular mass. This
protein was an unusual oligomerization state of a pro-
tein that was identified by immunoblotting as cN-II
but completely inactive. E. coli produces a small
amount of cN-II that is correctly folded and a large
amount of incorrectly folded and insoluble protein. It
is conceivable that a cN-II protein that is incorrectly

a
cN-II in 20 mM Tris ⁄ HCl, pH 8, +0.2 M NaCl (control)
(control), +5 m
M ATP 1.08 ± 0.08
(control), +5 m
M ⁄ 10 mM ATP ⁄ Mg
2+
1.16 ± 0.06
(control), +10 m
M ATP 1.13 ± 0.09
(control), +10 m
M ⁄ 10 mM ATP ⁄ Mg
2+
1.15 ± 0.08
(control), +5 m
M P
i
1.08 ± 0.07
(control), +5 m
M ⁄ 10 mM P
i
⁄ Mg
2+
0.95 ± 0.07
(control), +100 l
M AP
4
A 1.11 ± 0.10
a
Ratio of molecular mass in the presence of specific effectors to

method described by Fisher and Pei [18], and the point
mutants Y55G, T56V and H428D were produced as
described in the QuikChange
Ò
site-directed mutagenesis kit
manual (Stratagene, La Jolla, CA, USA). The primers used
are listed in Table 5.
Expression and purification of the recombinant
proteins
Expression of the recombinant mutants was performed as
previously described [19]. The 6· His-tagged proteins were
purified using the Ni-NTA agar method as described in the
QIAexpressionistÔ handbook (Qiagen). The protein con-
centration was determined using the Bradford method [20],
with BSA as the standard.
Enzyme assays
The nucleotidase activity of cN-II and its mutants was mea-
sured as the rate of [8-
14
C]-inosine formation from 2 mm
[8-
14
C]-IMP in the presence of 1.4 mm inosine, 20 mm
MgCl
2
, 4.5 mm ATP and 5 mm dithiothreitol, as previously
described [9]. Phosphotransferase activity was measured as
the rate of [8-
14
C]-IMP formation from 1.4 mm [8-

expected value of 2 was determined for the ratio between
nucleotidase and phosphotransferase activities under the
experimental conditions used for the wild-type recombinant
cN-II assay. Accordingly, an alteration of this ratio for a
mutant was considered as caused either by an alteration of
the K
m
value for one of the two substrates or by a variation
of the k
cat
value for one of the two activities. When
required, the rate of phosphate formation (phosphatase
activity) was measured as described by Chifflet et al. [21].
One unit of enzyme activity is the amount of enzyme
Table 5. Primers used for site-directed mutagenesis.
Mutant Forward primer (5¢ to 3¢) Reverse primer (5¢ to 3¢)
F36R GCGCGTGAACCGGAGTT ACCCGATGATAGGCTTC
Y115A CGCTGGAAACCTCTTGG GCATCAACTTTCAAAAGAT
F127E CGAGATAAGGGGACCAG TTAAATCCATGTGCACAG
R144E AGAAGATGACACTGAAAG TGAATAAATTTATTTGGATAC
I152D CGATCTGAACACACTATTC TAAAATCTTTCAGTGTCAT
N154D GGACACACTATTCAACCT AGAATGTAAAATCTTTCAGT
K311A CGCGCTGAAAATTGGTAC CCAGTTTTAGTATCCACC
G319D GGACCCCTTACAGCA GTGTAGGTACCAATTTTC
D396A GGCTATTTTCTTGGCTGA AAGCTCTGAAGCTCTTC
M436W GTGGATGGGGAGCCTG CCGTAGCACATGTCCA
H428D AAGAAAGTAACTGACGACATGGACATGTG CACATGTCCATGTCGTCAGTTACTTTCTT
Y55G AGTGTTTTGGGTTTGACATGGATGGCACACTTGCTG CAGCAAGTGTGCCATCCATGTCAAACCCAAAACACT
T56V AGTGTTTTGGGTTTGACATGGATTATGTGCTTGCTG CAGCAAGCACATAATCCATGTCAAACCCAAAACACT
Regulation of cytosolic 5¢-nucleotidase R. Pesi et al.

same protein concentration and refractive index. Under
these conditions, their ratio, according to Parr and Ham-
mes [22], is proportional to the ratio of molecular mass.
Acknowledgements
We would like to thank Dr Giovanni Strambini and
Dr Margherita Gonelli of the Institute of Biophysics
(National Research Centre, Pisa, Italy) for the light-
scattering analysis. We would also like to thank Dr
Adrian Wallwork for careful language revision of the
manuscript. This work was supported by a grant from
the Ministero dell’Istruzione, dell’Universita
`
e della
Ricerca and by local funds from the University of
Pisa.
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