Insights into the interaction of human arginase II with
substrate and manganese ions by site-directed
mutagenesis and kinetic studies
Alteration of substrate specificity by replacement of Asn149
with Asp
Vasthi Lo
´
pez, Ricardo Alarco
´
n, Marı
´
a S. Orellana, Paula Enrı
´
quez, Elena Uribe, Jose
´
Martı
´
nez and
Nelson Carvajal
Departamento de Bioquı
´
mica y Biologı
´
a Molecular, Facultad de Ciencias Biolo
´
gicas, Universidad de Concepcio
´
n, Chile
Arginase (l-arginine urea amidino hydrolase,
EC 3.5.3.1) catalyzes the hydrolysis of l-arginine to
yield l-ornithine and urea, and exhibits an absolute

Biologı
´
a Molecular, Facultad de Ciencias
Biolo
´
gicas, Universidad de Concepcio
´
n,
Casilla 160-C, Concepcio
´
n, Chile
Fax: +56 41 239687
E-mail:
(Received 24 May 2005, revised 13 July
2005, accepted 19 July 2005)
doi:10.1111/j.1742-4658.2005.04874.x
To examine the interaction of human arginase II (EC 3.5.3.1) with sub-
strate and manganese ions, the His120Asn, His145Asn and Asn149Asp
mutations were introduced separately. About 53% and 95% of wild-type
arginase activity were expressed by fully manganese activated species of the
His120Asn and His145Asn variants, respectively. The K
m
for arginine (1.4–
1.6 mm) was not altered and the wild-type and mutant enzymes were essen-
tially inactive on agmatine. In contrast, the Asn149Asp mutant expressed
almost undetectable activity on arginine, but significant activity on agma-
tine. The agmatinase activity of Asn149Asp (K
m
¼ 2.5 ± 0.2 mm) was
markedly resistant to inhibition by arginine. After dialysis against EDTA,

Asn149fiAsp mutation is proposed to generate a conformational change
responsible for the altered substrate specificity of arginase II. We also con-
clude that, in contrast with arginase I, Mn
2+
A
is the more tightly bound
metal ion in arginase II.
4540 FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS
substrate l-arginine [11,13]. Particularly interesting has
been a possible role of arginase II in regulating the
availability of l-arginine for nitric oxide synthesis in
human penile and clitoral corpus cavernosum and
vagina, which converts this isoenzyme in a potential
target for the treatment of sexual arousal disorders
[9,14,15].
At present, there is considerable information about
the structural and functional properties of arginase I,
whose deficiency in humans results in hyperarginine-
mia, characterized by growth retardation and progres-
sive mental impairment [8]. Although significantly less
is known about arginase II, the enzyme has been
cloned [16–18], some of their kinetic properties have
been described [9–12] and the X-ray crystal structure
of a fully active, truncated form complexed with a
boronic acid transition state analog inhibitor was
determined at 2.7 A
˚
resolution [9].
Human arginases I and II are related by about 50%
amino acid sequence identity and, more importantly,

The metal cluster of human arginase II was found
to be nearly identical to that of rat liver arginase I in
its complex with the transition state analog S-(2-boro-
noethyl)-l-cysteine (BEC). His120 and His145, and the
corresponding His101 and His126 in arginase I, were
described among the ligands for coordination of
Mn
2+
A
and Mn
2+
B
, respectively [9,24]. However, the
volume of the active site cleft was found to be larger
for arginase II. Moreover, the D232 (Od1)-Mn
2+
B
separation of 2.6 A
˚
was considered to be somewhat
long for an inner-sphere coordination interaction, as
that observed in arginase I [9]. Differences in the bind-
ing of the a-carboxylate and a-amino groups of BEC
were also ascribed to the larger volume of the active
site cleft of arginase II, which allows more water-medi-
ated enzyme–inhibitor interactions in this enzyme. For
example, Asn130 was identified as a ligand for the
a-carboxylate group of BEC in arginase I, but a water-
mediated hydrogen bond was proposed in place of a
direct hydrogen bond to the equivalent Asn149 in the

of arginase II were active even in the absence of added
Mn
2+
, although preincubation with 5 mm Mn
2+
for
10 min at 60 °C was required to convert the enzymes
to their fully active state. In contrast, the arginase
activity of the Asn149Asp variant was practically
undetectable, both before and after the incubation with
the manganese ions.
Fully active His120Asn and His145Asn variants
exhibited about 53% and 95% of the corresponding
wild-type activity, with the K
m
value for l-arginine
remaining essentially unaltered (Table 1). Considering
His120 and His145 as metal ligands in arginase II [9],
the essentially invariant K
m
value upon mutation of
these residues agree with the currently accepted mech-
anism for the arginase reaction, which considers the
V. Lo
´
pez et al. Interaction of arginase II with substrate and manganese ions
FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS 4541
metal ion as being involved in the stabilization of the
transition state [27], but not in the stabilization of the
substrate in the Michaelis–Menten complex [24]. Also

To further examine the effects of the His120fiAsn
and His145fiAsn mutations on the interaction of the
enzyme with manganese ions, maximally activated spe-
cies of the wild-type and mutant enzymes were dia-
lysed for 2 h at 4 °C against 10 mm EDTA in 10 mm
Tris ⁄ HCl pH 7.5, followed by two changes of the same
buffer but without EDTA. The dialyzed enzymes were
then assayed for catalytic activity and metal content
by atomic absorption analysis. As shown in Fig. 2,
after incubation with 5 mm Mn
2+
for 10 min at 60 °C
and assay in the presence of added 2 mm Mn
2+
, all of
the enzymes were active and measured activities were
essentially equal to the initial activity of the corres-
ponding fully activated control. However, when the
preincubation step was omitted and the assays were
performed in the absence of added Mn
2+
, the
His120Asn variant was found to be totally inactive,
whereas half of full activity was expressed by the
His145Asn mutant and wild-type enzymes. In agree-
ment with the inactive state of dialyzed species of the
His120Asn variant, its manganese content was almost
undetectable (< 0.1 Mn
2+
Æsubunit

Fig. 1. Fluorescence spectra (A) and sensitivity to thermal inactiva-
tion (B) of wild-type (s), H120N (d), H145N (h) and N149D (,)var-
iants of human arginase II. Fluorescence spectra were recorded at
25 °C; protein concentrations were 73, 82, 59 and 79 lgÆmL
)1
, for
the wild-type, His120Asn, His145Asn and Asn149Asp variants,
respectively. The line in (B) is for the average of experimental
values for all the enzyme variants.
Interaction of arginase II with substrate and manganese ions V. Lo
´
pez et al.
4542 FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS
contained 1.1 ± 0.1 Mn
2+
Æsubunit
)1
and 1.2 ± 0.1
Mn
2+
Æsubunit
)1
, respectively. Considerably more dras-
tic conditions were necessary to obtain metal-free,
inactive species of the wild-type and His145Asn
enzyme variants. Routinely, this was performed by
incubation for 1 h at 25 °C with 25 mm EDTA and
3 m guanidinium chloride in 10 mm Tris ⁄ HCl pH 7.5,
followed by overnight dialysis at 4 °C against 5 mm
Tris ⁄ HCl pH 7.5.

sidering the stoichiometry of 2 Mn
2+
Æsubunit
)1
derived
from EPR analysis of fully active arginase II [10], our
conclusion is that a weakly bound Mn
2+
is removed
by EDTA during the preparation of the samples for
atomic absorption analysis of the wild-type and
His145Asn variants. In addition to removal of the
more weakly bound Mn
2+
, the lower affinity for that
more tightly bound to the protein may explain the
absence of Mn
2+
from the EDTA-treated species of
the His145Asn variant. Even though under our condi-
tions the wild-type and His145Asn variants behaved
essentially in the same manner and expressed practi-
cally the same catalytic activity, an effect of the
His145Asn mutation on the affinity for the more
weakly bound metal ion cannot be discarded. The
presence of tightly and weakly bound manganese ions
was also demonstrated for fully active species of argi-
nase I [1,2]. Moreover, hyperbolic kinetics with dissoci-
ation constants for the more tightly bound Mn
2+

)1
, and metal-free, inactive
species of the His126Asn variant [31]. Our conclusion
is that the more weakly bound metal ion, which is
preferentially removed by EDTA, is Mn
2+
A
in argi-
nase I and Mn
2+
B
in arginase II. Because ligands to
the metal ions are strictly conserved in these enzymes,
the difference would reside in the length of the ligand–
metal separations. In this connection, the Asp232
(Od1)-Mn
2+
B
separation of 2.6 A
˚
in the arginase II–
BEC complex was considered somewhat long for an
inner-sphere coordination interaction, as that observed
in arginase I [9]. A lengthened His124 (Nd1)–Mn
2+
B
bond was also associated to the preferential release of
Mn
2+
B

FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS 4543
lower pK
a
for a water molecule bound to a binuclear
metal cluster and, consequently, by a higher concentra-
tion of the nucleophilic metal-bound hydroxide [33–35]
and increased stabilization of the transition state affor-
ded by the more weakly bound metal ion [27].
Altered substrate specificity accompanying the
Asn149Asp mutation
Interestingly, while essentially inactive on l-arginine,
the An149Asp variant exhibited a significant activity on
its decarboxylated derivative, agmatine (Fig. 3). The
possibility of interference from the endogenous agma-
tinase of the bacterial vector was excluded by the
DEAE-cellulose chromatographic step of the purifica-
tion protocol. In fact, like for all the arginase variants
examined in this study, 0.10–0.15 m KCl was required
to elute the Asn149Asp variant from a DEAE-cellulose
column equilibrated with 5 mm Tris ⁄ HCl pH 7.5,
whereas about 0.45 m KCl was required for elution of
the endogenous bacterial agmatinase. Moreover, the
arginase variants, including Asn149Asp, were not detec-
ted by Western blot analysis using an anti-Escherichia
coli agmatinase polyclonal antibody. Finally, in contrast
with E. coli agmatinase, the Asn149Asp was markedly
resistant to inhibition by arginine (Fig. 4).
Clearly, arginine was very poorly recognized as a
substrate or inhibitor by the Asn149Asp variant. The
opposite occurred with the wild-type, His120Asn and

type enzyme. As measured by k
cat
⁄ K
m
, the catalytic
efficiency of the Asn149Asp variant was found to be
about 36-fold lower than that of wild-type arginase II
acting on arginine. For comparison, the catalytic effi-
ciency of E. coli agmatinase [36] is only twofold lower
than that corresponding to wild-type arginase II. At
this connection, residues known to be involved in
binding and hydrolysis of the guanidino group of
l-arginine by arginase are strictly conserved in the act-
ive site of the agmatinases [19]. Moreover, modeling
studies have revealed that essentially the same position
with respect to the metal ions and conserved catalyti-
Fig. 3. Catalytic activity of the Asn149Asp variant of human argi-
nase II. Substrates were agmatine (s) and
L-arginine (d). The buf-
fer was 50 m
M glycine ⁄ NaOH pH 9.0.
Fig. 4. Effect of L-arginine on agmatine hydrolysis by the N149D
variant of arginase II (s)andE. coli agmatinase (d). The buffer was
50 m
M glycine ⁄ NaOH pH 9.0.
Interaction of arginase II with substrate and manganese ions V. Lo
´
pez et al.
4544 FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS
cally important residues may be adopted by agmatine

for its binding and hydrolysis by the Asn149Asp vari-
ant. However, if the only change were in the charge at
position 149, it would hard to explain why agmatine
not only is practically not hydrolysed by wild-type
arginase II, but it is also very poorly inhibitory to this
enzyme form. Thus, the altered specificity most prob-
ably reflect an active site conformational change
resulting from the Asn149fiAsp substitution. As
deduced from the unaltered fluorescence properties,
thermal stability and chromatographic behavior, the
conformational change is not expected to be extensive
enough to cause gross alterations in the enzyme struc-
ture. Studies addressed to further define the expected
conformational change, using experimental and com-
putational methods, will be initiated soon in our
laboratory.
General conclusions
In addition to substantiate the participation of His120
and His145 as ligands for the manganese ions in
human arginase II, our results have provided addi-
tional evidence for the differences between the active
sites of this enzyme and arginase I. In spite of the rel-
atively low agmatinase activity of the Asn149Asp vari-
ant, it is clear that the interactions of arginase II with
l-arginine and agmatine are greatly altered by replace-
ment of this residue with aspartate. To the best of our
knowledge, this is the first report in which the sub-
strate specificity of arginase was altered by using site-
directed mutagenesis.
Experimental procedures

2
and dialyzed for 6 h at
4 °C against the same buffer. After incubation with 5 mm
MnCl
2
for 10 min at 60 °C, the enzyme solution was separ-
ated by chromatography on a CM-cellulose column equili-
brated with 5 mm Tris ⁄ HCl pH 7.5; active fractions, eluting
with the washings of the column, were then chromato-
graphed on a DEAE-cellulose column equilibrated with
5mm Tris ⁄ HCl pH 7.5. Active fractions, eluting at 0.10–
0.15 m KCl, were pooled and dialyzed against 5 mm
Tris ⁄ HCl pH 7.5 containing 2 mm MnCl
2
. A single protein
band was detected by SDS ⁄ PAGE and Coomassie blue
staining of purified enzymes.
Metal-free species of purified enzymes were obtained by
incubation for 1 h at 25 °C with 25 mm EDTA and 3 m
V. Lo
´
pez et al. Interaction of arginase II with substrate and manganese ions
FEBS Journal 272 (2005) 4540–4548 ª 2005 FEBS 4545
guanidinium chloride in 10 mm Tris ⁄ HCl pH 7.5, followed
by overnight dialysis at 4 °C against 5 mm Tris ⁄ HCl
pH 7.5.
Site-directed mutagenesis
The His120Asn, His145Asn and Asn149Asp mutant forms of
human arginase II were obtained by a two-step PCR [40],
using the QuickChange site-directed mutagenesis kit (Strata-

.
Atomic absorption analysis
The manganese contents of arginase preparations were deter-
mined by atomic absorption on a Perkin Elmer 1100 atomic
absorption spectrometer (NY, USA) equipped with a
graphite furnace and a deuterium arc background corrector.
Recovery was nearly 100%. For analysis, the purified enzyme
was activated by incubation with 2 mm MnCl
2
in 10 mm
Tris ⁄ HCl pH 8.0 for 30 min at 37 °C, and then the free metal
ion was removed by dialysis against 10 mm Tris ⁄ HCl pH 7.5,
10 mm EDTA for 2 h at 4 °C, followed by two changes of
10 mm Tris ⁄ HCl pH 7.5 as the dialysis buffer.
Enzyme assays and kinetic studies
Routinely, enzyme activities were determined by measuring
the formation of urea from l-arginine or agmatine in
50 mm glycine ⁄ NaOH pH 9.0. In studying the effect of
pH on enzyme activities, buffers used were 50 mm
Tris ⁄ HCl pH 7–8.7 and 50 mm glycine ⁄ NaOH pH 8.7–10.
Urea was determined by a colorimetric method with a-iso-
nitrosopropiophenone [41]. As urea is also produced by
agmatine hydrolysis, in studying the inhibitory effect of
agmatine on arginine hydrolysis, reactions were followed
by measuring the formation of ornithine, determined by
the method of Chinard [42]. Protein concentrations were
determined by the method of Bradford [43], with BSA as
standard.
Steady-state initial velocity studies were performed at
37 °C and all assays were initiated by adding the enzyme

FONDECYT and Grant CONICYT to support the
PhD thesis of V. Lo
´
pez.
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