Báo cáo khoa học: Mutational analysis of substrate recognition by human arginase type I ) agmatinase activity of the N130D variant pot

Mutational analysis of substrate recognition by human
arginase type I
)
agmatinase activity of the N130D variant
Ricardo Alarco
´
n, Marı
´a
S. Orellana, Benita Neira, Elena Uribe, Jose
´
R. Garcı
´a
and Nelson Carvajal
Departamento de Bioquı
´
mica y Biologı
´
a Molecular, Facultad de Ciencias Biolo
´
gicas, Universidad de Concepcio
´
n, Chile
Arginase (l-arginine amidino hydrolase, EC 3.5.3.1)
catalyzes the hydrolysis of l-arginine to the products
l-ornithine and urea. The enzyme is widely distributed
and has several functions, including ureagenesis and
regulation of the cellular levels of arginine, a precursor
for the production of creatine, proline, polyamines and
the cell-signaling molecule NO [1–5]. Mammalian tissues
contain two distinct isoenzymic forms: arginase I, highly
expressed in the liver and traditionally associated with

ureagenesis, and the extrahepatic arginase II, which is
thought to provide a supply of ornithine for proline and
polyamine biosynthesis [1,3]. By competing with NO
synthases for a common substrate, arginase can effect-
ively regulate NO-dependent processes [1,4,6,7]. Thus,
arginase inhibitors have therapeutic potential in treating
NO-dependent smooth muscle disorders [8,9].
Closely related to the arginase reaction is the hydro-
lysis of agmatine to putrescine and urea, catalyzed by
agmatinase (agmatine amidinohydrolase, EC 3.5.3.11).
Agmatine (1-amino-4-guanidinobutane), a primary
amine that results from decarboxylation of arginine by
arginine decarboxylase, is an intermediate in polyamine
biosynthesis [8,10], and may have important regulatory
roles in mammals, including neurotransmitter ⁄ neuro-
modulatory actions [11,12] and regulation of hepatic
ureagenesis [13]. Like arginase, agmatinase is absolutely
dependent on manganese ions [14], which are thought to
participate in the activation of a water molecule to gen-
erate a metal-bound hydroxide that nucleophilically
attacks the guanidino carbon of the corresponding sub-
strate [14–16]. Moreover, residues involved in metal
binding and substrate hydrolysis are strictly conserved
among these two enzymes [11,12,17–20]. However, argi-
nase and agmatinase are highly discriminatory between
arginine and its decarboxylated derivative. Thus, the
k
cat
⁄ K
m

value is reduced about 50 000-fold when argin-
ine is replaced by agmatine as the substrate for rat liver
arginase [21], human arginase II is practically inactive
on agmatine [22], and agmatinase does not utilize argin-
ine as a substrate [22]. One important question to ask
concerns, therefore, the key structural determinants
of substrate discrimination by these two enzymes,
which are considered to have a common evolutionary
origin [18].
An important insight into the molecular basis of
substrate binding to arginase was provided by the
Keywords
agmatine; arginase; asparagine 130; human
liver; substrate specificity
Correspondence
N. Carvajal, Departamento de Bioquı
´
mica y
Biologı
´
a Molecular, Facultad de Ciencias
Biolo
´
gicas, Universidad de Concepcio
´
n,
Casilla 160-C, Concepcio
´
n, Chile
Fax: +56 41 239687

Tel: +56 41 2204428
E-mail:
(Received 29 August 2006, revised 13
October 2006, accepted 23 October 2006)
doi:10.1111/j.1742-4658.2006.05551.x
Upon mutation of Asn130 to aspartate, the catalytic activity of human
arginase I was reduced to  17% of wild-type activity, the K
m
value for
arginine was increased  9-fold, and the k
cat
⁄ K
m
value was reduced  50-
fold. The kinetic properties were much less affected by replacement of
Asn130 with glutamine. In contrast with the wild-type and N130Q
enzymes, the N130D variant was active not only on arginine but also on
its decarboxylated derivative, agmatine. Moreover, it exhibited no preferen-
tial substrate specificity for arginine over agmatine (k
cat
⁄ K
m
values of
2.48 · 10
3
m
)1
Æs
)1
and 2.14 · 10

3
m
)1
Æs
)1
, respectively). After dialysis
against EDTA and assay in the absence of added Mn
2+
, the N130D
mutant enzyme was inactive, whereas about 50% full activity was
expressed by the wild-type and N130Q variants. Mutations were not
accompanied by changes in the tryptophan fluorescence properties, thermal
stability or chromatographic behavior of the enzyme. An active site con-
formational change is proposed as an explanation for the altered substrate
specificity and low catalytic efficiency of the N130D variant.
FEBS Journal 273 (2006) 5625–5631 ª 2006 The Authors Journal compilation ª 2006 FEBS 5625
structures of the rat and human liver enzymes com-
plexed with transition state analogs [9,23,24] and the
binary complex of Bacillus caldovelox arginase with
arginine [25]. From these studies, the a-carboxylate
group of arginine appears to be hydrogen bonded to
the N130 carboxamide NH
2
group and the S137
hydroxyl group of arginase I [9,23,24]. These residues
and those considered as ligands for the substrate
a-amino group are highly conserved in essentially all
the arginases [17]. Exceptions are the plant arginases,
although these enzymes are also specific for arginine
over agmatine and, like the nonplant arginases, they

are competitively inhibited by N
G
-hydroxy-l-arginine,
an intermediate of NO synthesis [26]. On the other
hand, in contrast with Asn130 in arginase I, the corres-
ponding Asn149 of arginase II was not found to be a
ligand for the a-carboxylate group of the transition
state analog S-(2-boronoethyl)-l-cysteine [4]. Neverthe-
less, the arginase activity of arginase II is practically
lost when Asn149 is replaced with aspartate [22].
To get a better understanding of the importance of
Asn130 in substrate binding and recognition by argi-
nase I, this residue was changed to aspartate by site-
directed mutagenesis, and the kinetic consequences of
the mutation were examined. Special emphasis was
placed on the potential ability of the enzyme to utilize
agmatine as a substrate. In contrast with the corres-
ponding N149D variant of human arginase II, which
was found to be almost exclusively active on agmatine
[22], the N130D variant of arginase I exhibited no
preferential specificity for arginine over agmatine. In
addition to supporting a critical role for Asn130 in
arginase I, our present results further substantiate the
existence of significant differences between the active
sites of arginases I and II [22].
Results and Discussion
As shown in Table 1, upon mutation of Asn130 to
aspartate, the arginase activity of human arginase I was
reduced to about 17% of the wild-type activity, and the
K

m
value for arginine was increased about nine-fold.
The result was markedly decreased catalytic efficiency
of the N130D variant, as indicated by a 50-fold reduc-
tion in k
cat
⁄ K
m
value. There was also decreased affinity
of the N130D mutant enzyme for ornithine, whereas the
affinity for the dead-end inhibitor guanidinium chloride
was not substantially altered. Both inhibitions were
slope-linear competitive. As our activity assay is based
on determination of the urea produced by substrate
hydrolysis, it was not possible to perform product inhi-
bition studies with urea. However, considering the
structural analogies between guanidinium chloride and
urea, the natural product of arginine hydrolysis may be
reasonably expected to bind at the same site as the
dead-end analog, causing competitive inhibition. On
this basis, our results agree with a rapid equilibrium
random release of products from both enzyme variants.
The same kinetic mechanism was previously reported
for rat liver arginase [21] and also for agmatine hydro-
lysis by Escherichia coli agmatinase [31].
The Asn130 fi Asp mutation was also accompanied
by increased sensitivity of the enzyme to inhibition by
agmatine and putrescine, the decarboxylated deriva-
tives of arginine and ornithine, respectively (Fig. 1).
However, the most important difference was in the

ability of the N130D variant to utilize not only argin-
ine, but also agmatine, as a substrate (Fig. 2; Table 1).
Moreover, the mutant enzyme exhibited no preferential
substrate specificity for arginine over its decarboxylat-
ed derivative, as indicated by similar k
cat
⁄ K
m
values
(Table 1). Clearly, the lower k
cat
value for agmatine
hydrolysis was compensated by a lower K
m
value for
this substrate. In contrast with the N130D variant of
human arginase I, the K
m
value of rat liver arginase
for agmatine was reported to be about 10 times higher
than that for agmatine; reported values were
1 ± 0.1 mm and 10.8 ± 1.7 mm, respectively [21].
Putrescine, a product of agmatine hydrolysis, was a
linear competitive inhibitor of agmatine and arginine
hydrolysis by the N130D variant, with K
i
values of
1.3 ± 0.2 mm and 1.5 ± 0.1 mm, respectively. The
Table 1. Kinetic properties of the wild-type and N130D variants of human arginase I. The inhibitors used were ornithine (orn) and guanidi-
nium chloride (Gdn).

Enzyme
Substrate
Arginine Agmatine
k
cat
(s
)1
)
K
m
(mM)
k
cat
⁄ K
m
(M
)1
Æs
)1
)
K
i
orn
(mM)
K
Gdn
i
(mM)
k
cat

(s
)1
)
K
m
(mM)
k
cat
⁄ K
m
(M
)1
Æs
)1
)
Wild-type 190 ± 10 1.5 ± 0.2 1.27 · 10
5
1.0 ± 0.2 56 ± 4 – – –
N130D 33 ± 5 13.3 ± 2.5 2.48 · 10
3
7.8 ± 0.1 39 ± 3 3 ± 1 1.4 ± 0.3 2.14 · 10
3
Agmatinase activity of the N130D mutant arginase I R. Alarco
´
n et al.
5626 FEBS Journal 273 (2006) 5625–5631 ª 2006 The Authors Journal compilation ª 2006 FEBS
coincident K
i
values are in agreement with the same
enzyme–putrescine complex being involved in both

competitive inhibitions. Also as expected for reactions
catalyzed by the same molecular entity, the thermal
inactivations of arginase and agmatinase activities of
the N130D variant were totally coincident (Fig. 3A).
Any interference from the endogenous agmatinase
activity of the bacterial strain used to express the
recombinant enzymes was excluded by the DEAE–
cellulose chromatographic step of the purification
protocol and the immunological properties of the argi-
nase variant. In fact, wild-type and N130D arginases
were not retained by a DEAE–cellulose column equili-
brated with 5 mm Tris ⁄ HCl (pH 7.5), whereas about
0.45 m KCl was required for elution of the endogenous
bacterial agmatinase. On the other hand, like the wild-
type enzyme, the N130D mutant was not recognized
by an antibody to E. coli agmatinase (Fig. 3B).
The wild-type and N130D variants were also differ-
ently affected by dialysis against EDTA. As shown in
Fig. 1. Inhibition of wild-type and N130D
variants of human arginase I by agmatine
(A) and putrescine (B). The arginine concen-
tration was 5 m
M and the assays were per-
formed at pH 9.0. Reactions were followed
by measuring the production of
L-ornithine
from
L-arginine. Initial velocities in the
absence and presence of putrescine are
expressed as v

o
and v
i
, respectively.
Fig. 2. Effects of varying concentrations of agmatine as a substrate
for the wild-type and N130D variants of human arginase I. Reac-
tions were followed by measuring the production of urea at pH 9.0.
Enzyme activities are expressed as lmol ureaÆmin
)1
.
A
B
Fig. 3. (A) Thermal inactivation of N130D. At the indicated times of
incubation at 80 °C, residual arginase (d) and agmatinase (s) activ-
ities were determined as described under Experimental procedures.
(B) Western blot analysis of wild-type (WT) and N130D variants of
human arginase I using an antibody raised against E. coli agma-
tinase (AUH). The relative molecular masses of the protein markers
(ST), ovoalbumin (42 700) and carboanhydrase (30 000) are indica-
ted by the numbers on the right side. The migration of the protein
markers is indicated by pencil marks on the membrane. The arrow
indicates the position of the wild-type and N130D variants of
human arginase, as detected by an antibody to human arginase I.
R. Alarco
´
n et al. Agmatinase activity of the N130D mutant arginase I
FEBS Journal 273 (2006) 5625–5631 ª 2006 The Authors Journal compilation ª 2006 FEBS 5627
Fig. 4, whereas dialyzed wild-type species expressed
about 50% of full arginase activity in the absence of
added Mn

2+
, the N130D variant became totally
dependent on added Mn
2+
for catalytic activity. In
any case, the initial activity of the corresponding fully
activated control was almost completely recovered
after preincubation and assay of dialyzed species in the
presence of 2 mm Mn
2+
. Confirming previous results,
considerably more drastic conditions were required to
convert wild-type arginase I to inactive, metal-free spe-
cies [32]. These conditions included preincubation with
10 mm EDTA followed by dialysis for at least 12 h at
4 °C. Thus, although Asn130 is not a ligand for metal
coordination in arginase [15], it is clear that the stabil-
ity of the Mn
2+
-binding site was altered by replace-
ment of this residue with aspartate, presumably
through a conformational change driven by the negat-
ive charge introduced at position 130.
The relevance of the negative charge at position 130
for the altered properties of the N130D variant was
supported by the effects of introducing a glutamine
residue at this position. In fact, the N130Q variant
was found to be totally inactive on agmatine, although
it retained about 60% of the wild-type arginase activ-
ity and exhibited a marginally increased K

m
value for
arginine ( 2-fold). On the other hand, upon dialysis
against EDTA, the N130Q variant behaved exactly as
described for the wild-type species.
The presumed conformational change accompanying
the Asn130 fi Asp mutation is most probably restric-
ted to the active site. Gross alterations may be reason-
ably discounted, considering the following findings:
(a) the environment of the tryptophan residues was
not substantially altered, as indicated by the main-
tenance of the tryptophan fluorescence properties of
the enzyme (k
max
¼ 340 nm); (b) under the conditions
used in this study (presence of 2 mm Mn
2+
,80°C and
pH 7.5), there was no significant difference in the
thermal stability of the wild-type and mutant enzymes;
and (c) the ion exchange chromatographic properties
on a DEAE–cellulose column and oligomeric structure
of the enzyme (molecular mass of 120 kDa) were
not altered. The tryptophan fluorescence proper-
ties, thermal stability and chromatographic behaviour
of the enzyme were also not altered by the
Asn130 fi Gln mutation.
Asn130 was proposed as a ligand for the a-carboxyl
group of arginine in arginase I [9,23,24]. On this basis,
direct repulsion of the negatively charged Asp130

would explain the increased K
m
value for arginine
inhibition and K
i
value for ornithine inhibition of the
N130D variant. The large effect of the Asn130 fi Asp
mutation on the k
cat
value indicates that arginine is
not correctly positioned for optimal nucleophilic
attack by the metal-bound hydroxide in the conforma-
tionally altered active site of the N130D variant. On
the other hand, as a potential ligand for a primary
amino group, Asp130 would contribute to the
increased affinity of the mutant enzyme for agmatine
and putrescine and, thus, to its ability to act on the
decarboxylated derivative of arginine. However, the
possibility of interaction with residues such as Asp193,
proposed as ligands for the a-amino group of arginine
in arginase I [9], cannot be discounted. Whatever the
case, the substantially low k
cat
value argues against
correct positioning of agmatine with respect to the
metal-bound hydroxide in the active site of the N130D
variant.
A previously reported homology-modeled structure
of E. coli agmatinase was found to be very similar to
those of the crystallographically defined rat liver and

B. caldovelox arginases [31] with respect to the number
and arrangement of structural elements ( a-helix and
b-sheets). One significant difference was the shorter
extension of a loop located at the entrance of the act-
ive site cleft. The arginase loop (residues 126–143 in
the sequence of human liver arginase) contains Asn130
and other residues proposed as ligands for the a-carb-
oxylate group of the substrate arginine. As this is the
Fig. 4. Effects of dialysis against EDTA on the catalytic activity of
wild-type and N130D variants of human arginase I. Fully active spe-
cies were dialysed for 2 h at 4 °C against 10 m
M EDTA in 10 mM
Tris ⁄ HCl (pH 7.5), and then for 2 h under the same conditions but
in the absence of EDTA. Dialyzed species were assayed for argi-
nase activity in the absence of added Mn
2+
(open bars) and after
full activation with the metal ion and assay in the presence of
added 2 m
M Mn
2+
(filled bars). Arginase activities are expressed as
percentage of the initial activity of the corresponding fully activated
species.
Agmatinase activity of the N130D mutant arginase I R. Alarco
´
n et al.
5628 FEBS Journal 273 (2006) 5625–5631 ª 2006 The Authors Journal compilation ª 2006 FEBS
part of the molecule that makes arginine different from
agmatine, our results support the concept that differ-

ences in this loop area are key factors in determining
the differences in substrate specificity between arginase
and agmatinase [31]. Interestingly, when compared
with rat liver and B. caldovelox arginases, the active
site of Deinococcus radiodurans agmatinase was found
to deviate mostly in three regions [33]. One of these
regions (the b6–a5 loop defined by residues 146–157),
considered as contributing to provision of the struc-
tural determinants for substrate specificity, is equival-
ent to the already described E. coli loop.
In conclusion, the results obtained indicate that both
the substrate specificity and catalytic efficiency of
human arginase I are markedly altered by replacement
of Asn130 with aspartate. In addition to supporting the
importance of Asn130 in substrate binding and discrim-
ination by arginase I, our present results further corro-
borate the existence of active site differences between
the isoenzymic forms of human arginase. In fact,
whereas partial arginase activity is retained by the
N130D variant of arginase I, the corresponding N149D
variant of arginase II was found to be active practically
only on agmatine [22]. This adds to the crystallographic
evidence for a larger volume of the active site cleft of
arginase II [4], and our previous report that Mn
2+
A
and
not Mn
2+
B

, as occurs in arginase I, is the more tightly
bound metal ion in human arginase II [22].
Experimental procedures
Materials
All reagents were of the highest quality commercially avail-
able (most from Sigma Chemical Co., St Louis, MO, USA)
and were used without further purification. Restriction
enzymes, as well as enzymes and reagents for PCR, were
obtained from Promega (Madison, WI, USA). The plasmid
pBluescript II KS(+), containing the human liver arginase
type I cDNA, and the antibody to arginase I, were kindly
provided by S. Cederbaum (University of California, Los
Angeles, CA, USA). The antibody raised against E. coli
agmatinase was provided by M. Salas (Universidad Austral,
Valdivia, Chile). The protein standard mixture IV (M
r
range 12 300–78 000) was obtained from Merck (Darms-
tadt, Germany). Horseradish peroxidase-labeled anti-rabbit
IgG and LumiGlo chemiluminiscent substrate were pur-
chased from KPL (Gaithersburg, MD, USA).
Enzyme preparations
Bacteria were grown with shaking at 37 °C in Luria broth
in the presence of ampicillin (100 lgÆmL
)1
). The wild-type
and mutant human liver arginase cDNAs were directional-
ly cloned into the pBluescript II K(+) E. coli expression
vector and expressed in E. coli strain JM109, following
induction with 1 mm isopropyl thio-b-d-thiogalactoside.
The bacterial cells were disrupted by sonication on ice

(5 · 30 s pulses) and the supernatant resulting from centrif-
ugation for 20 min at 12 000 g (Sorval RC5C Plus centri-
fuge with SS-34 rotor) was precipitated with ammonium
sulfate (60% saturation). The pellet, recollected by centrifu-
gation at 12 000 g for 10 min (Sorval RC5C Plus centrifuge
with SS-34 rotor), was resuspended in 5 mm Tris ⁄ HCl
(pH 7.5) containing 2 mm MnCl
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 variants were puri-
fied as previously described [23]. The purity of the enzymes
was evaluated by SDS ⁄ PAGE. In SDS ⁄ PAGE, the wild-
type and mutant forms comigrated, as determined by stain-
ing with Coomassie Brilliant Blue R250 and western blot-
ting using an antibody to human liver arginase.
Site-directed mutagenesis
The N130D and N130Q mutant forms were obtained by a
two-step PCR [27], using the plasmid pBluescript II KS(+)
containing the human liver arginase cDNA as a template
and the QuickChange site-directed mutagenesis kit (Strata-
gene, La Jolla, CA, USA). The antisense and sense muta-
genic oligonucleotide primers for the N130D variant were
5¢-GTGGAGTGTCGATATCA-3¢ and 5¢-TGATATCGAC
ACTCCAC-3¢, respectively. The corresponding primers for
the Asn130 fi Gln mutation were 5¢-GTGGAGTTTGGA
TATCA-3¢ and 5¢-TGATATCCAAACTCCAC-3¢.
The expected mutations were confirmed by DNA

sequence analysis. That no unwanted mutations had been
introduced during the mutagenesis process was also con-
firmed by automated sequence analysis.
Enzyme assays and kinetic studies
Routinely, enzyme activities were determined by measuring
the production of urea from l-arginine or agmatine in
50 mm glycine ⁄ NaOH (pH 9.0) at 37 °C. As urea is also a
product of agmatine hydrolysis, arginase activities in the
presence of agmatine were assayed by measuring the
production of l-ornithine. Urea was determined by a
colorimetric method with a-isonitrosopropiophenone [28],
and l-ornithine by the method of Chinard [29]. Protein
concentrations were estimated by the method of Bradford
[30], with BSA as standard.
To examine the stability of the enzyme–metal interac-
tion, the enzymes were dialyzed for for 2 h at 4 °C
against 10 mm EDTA in 5 mm Tris ⁄ HCl (pH 7.5) and
then for 2 h in the absence of the chelating agent. Dia-
lyzed species were assayed for arginase activity both in
R. Alarco
´
n et al. Agmatinase activity of the N130D mutant arginase I
FEBS Journal 273 (2006) 5625–5631 ª 2006 The Authors Journal compilation ª 2006 FEBS 5629
the absence of added Mn
2+
and after incubation with
2mm Mn
2+
for 10 min at 60 °C.
Steady-state initial velocity studies were performed at

37 °C, and all assays were initiated by adding the enzyme to
a previously equilibrated buffer substrate solution. The
enzymes had been previously incubated with 2 mm Mn
2+
for
10 min at 60 °C, and all the assays were performed in the
presence of added 2 mm Mn
2+
. Data from initial velocity
and inhibition studies, performed in duplicate and repeated
at least three times, were fitted to the appropriate equations,
by using nonlinear regression with prism 4.0 (GraphPad
Software Inc., San Diego, CA, USA). The standard errors of
the estimates were less than 6–7% of mean values.
Fluorescence spectra and thermal inactivation
studies
Fluorescence measurements were performed at 25 °Cona
Shimadzu (Kyoto, Japan) RF-5301 spectrofluorimeter. The
protein concentration was 40–50 lgÆmL
)1
, and emission
spectra were measured with an excitation wavelength of
295 nm. The slit width for both excitation and emission
was 1.5 nm, and spectra were corrected by subtracting the
spectrum of the buffer solution (5 mm Tris ⁄ HCl, pH 7.5) in
the absence of protein.
The stability to thermal inactivation was examined by
incubation of the enzymes at 80 °C in a solution containing
10 mm Tris ⁄ HCl (pH 7.5) and 2 mm Mn
2+

. At several time
points, aliquots were removed and assayed for residual cat-
alytic activity at pH 9.0, in the presence of added 2 mm
Mn
2+
.
Western blot analysis
Protein samples were electrophoresed on 12% SDS–poly-
acrylamide gels and then electroblotted onto a nitrocellu-
lose membrane at 200 mA for 3 h in a buffer solution
(pH 12) containing 25 mm Tris ⁄ HCl, 192 mm glycine, and
20% methanol. The membrane was blocked with TBS-
Tween (Tris-buffered saline containing 0.05% Tween-20)
and 0.5% skimmed milk for 1 h at room temperature, and
this was followed by incubation for 1 h at 4 °C with anti-
bodies raised against human arginase I or E. coli agma-
tinase. After washings with TBS-Tween, the membrane was
allowed to bind horseradish peroxidase-labeled anti-(rabbit
IgG) diluted 1 : 5000 in TBS-Tween for 1 h at room tem-
perature. This was followed by washings with TBS-Tween
and detection with the LumiGlo chemiluminiscent reagent
(KPL).
Acknowledgements
This research was supported by Grant 1030038 from
FONDECYT.
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