Thus, the kcat⁄ Kmvalue is reduced about 50 000-fold when argin-ine is replaced by agmatargin-ine as the substrate for rat liver arginase [21], human arginase II is practically inactive
Trang 1arginase 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 agmatiargi-nase are highly discriminatory between arginine and its decarboxylated derivative Thus, the
kcat⁄ Kmvalue is reduced about 50 000-fold when argin-ine is replaced by agmatargin-ine 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: ncarvaja@udec.cl
(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 Km value for arginine was increased 9-fold, and the kcat⁄ Km 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 (kcat⁄ Km values of 2.48· 103m)1Æs)1 and 2.14· 103m)1Æs)1, respectively) After dialysis against EDTA and assay in the absence of added Mn2+, 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
Trang 2structures 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 NH2 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 NG-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
Km 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 kcat⁄ Kmvalue 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 Asn130fi 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 agmatargin-ine, as a substrate (Fig 2; Table 1) Moreover, the mutant enzyme exhibited no preferential substrate specificity for arginine over its
decarboxylat-ed derivative, as indicatdecarboxylat-ed by similar kcat⁄ Km values (Table 1) Clearly, the lower kcat value for agmatine hydrolysis was compensated by a lower Km value for this substrate In contrast with the N130D variant of human arginase I, the Km 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 Ki 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
k cat
(s)1)
K m
(m M )
k cat ⁄ K m
( M )1Æs)1)
K iorn
(m M )
KGdni (m M )
k cat
(s)1)
K m
(m M )
k cat ⁄ K m
( M )1Æs)1)
Wild-type 190 ± 10 1.5 ± 0.2 1.27 · 10 5
Trang 3coincident Ki 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 argiargi-nases
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.
Trang 4Fig 4, whereas dialyzed wild-type species expressed
about 50% of full arginase activity in the absence of
added Mn2+, the N130D variant became totally
dependent on added Mn2+ 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 Mn2+ 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
4C Thus, although Asn130 is not a ligand for metal
coordination in arginase [15], it is clear that the
stabil-ity of the Mn2+-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 Km 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 Asn130fi 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 (kmax¼ 340 nm); (b) under the conditions used in this study (presence of 2 mm Mn2+, 80C 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 Asn130fi 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 Km value for arginine inhibition and Ki value for ornithine inhibition of the N130D variant The large effect of the Asn130fi Asp mutation on the kcat 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 kcat 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 caldoveloxarginases [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 m M
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.
Trang 5part 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 Mn2+Aand
not Mn2+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 (Mr
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 37C 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 MnCl2 and dialyzed for 6 h at
4C against the same buffer After incubation with 5 mm MnCl2for 10 min at 60C, 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 Asn130fi 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 4C 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
Trang 6the absence of added Mn2+ and after incubation with
2 mm Mn2+ for 10 min at 60C
Steady-state initial velocity studies were performed at
37C, 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 Mn2+for
10 min at 60C, and all the assays were performed in the
presence of added 2 mm Mn2+ 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 25C on a
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 80C in a solution containing
10 mm Tris⁄ HCl (pH 7.5) and 2 mm Mn2+
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
Mn2+
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% TBS-Tween-20)
and 0.5% skimmed milk for 1 h at room temperature, and
this was followed by incubation for 1 h at 4C 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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