Tài liệu Báo cáo khoa học: 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 docx

About 53% and 95% of wild-type arginase activity were expressed by fully manganese activated species of the His120Asn and His145Asn variants, respectively.. The Asn149fiAsp mutation alter

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

requirement for bivalent metal ions, especially Mn2+,

for catalytic activity Metal ions are thought to

acti-vate a coordinated water molecule, by lowering the

pKa for proton ionization and generation of the

hydroxide that nucleophilically attacks the guanidino

carbon of the scissile bond of l-arginine [1–3]

The enzyme is widely distributed in living organisms,

where it serves several functions, including ureagenesis

and regulation of the cellular levels of l-arginine, a precursor for the production of creatine, proline, poly-amines and nitric oxide [4–7] Mammalian tissues con-tain two distinct isoenzymic forms: arginase I, which is highly expressed in the liver and it has been tradition-ally associated with ureagenesis, and the extrahepatic arginase II, which is thought to provide a supply of

l-ornithine for proline and polyamine biosynthesis [7–12] Both arginase isoforms are also thought to par-ticipate in the regulation of nitric oxide biosynthesis by competing with nitric oxide synthases for the common

Keywords

manganese ions; histidine; agmatinase

activity; arginase II; human

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

E-mail: ncarvaja@udec.cl

(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 Kmfor 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 (Km¼ 2.5 ± 0.2 mm) was markedly resistant to inhibition by arginine After dialysis against EDTA, the His120Asn variant was totally inactive in the absence of added Mn2+ and contained < 0.1 Mn2+Æsubunit)1, whereas wild-type and His145Asn enzymes were half active and contained 1.1 ± 0.1 Mn2+Æsubunit)1 and 1.3 ± 0.1 Mn2+Æsubunit)1, respectively Manganese reactivation of metal-free to half active species followed hyperbolic kinetics with Kd of 1.8 ± 0.2· 10)8m for the wild-type and His145Asn enzymes and 16.2 ± 0.5· 10)8m for the His120Asn variant Upon mutation, the chro-matographic behavior, tryptophan fluorescence properties (kmax¼ 338–

339 nm) and sensitivity to thermal inactivation were not altered The 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, Mn2+A is the more tightly bound metal ion in arginase II

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,

residues which are known to be involved in metal

coordination, substrate binding and catalysis are

strictly conserved between the two isoenzymes [9]

Moreover, a binuclear metal cluster (Mn2+A–Mn2+B)

is accepted to be required for maximal catalysis by

both enzyme forms [9] Mammalian and all other

known arginases also shares a significant sequence

homology with all sequenced agmatinases (agmatine

ureo hydrolase, EC 3.5.3.11), which catalyzes the

hydrolytic production of urea from agmatine, a

decar-boxylated derivative of arginine [19–21] In view of

this, a common evolutionary origin and subsequent

divergence, resulting in totally different substrate

spe-cificities, is considered for arginase and agmatinase

[20] Such substrate discrimination is particularly

important for mammalian arginase II and agmatinase,

as both enzymes are mitochondrial and functionally

different [22] A key factor is the a-carboxyl group of

the substrate, which makes the difference between

arginine and agmatine Agmatine, which results from

decarboxylation of arginine by arginine decarboxylase,

is a metabolic intermediate in the biosynthesis of

putrescine and higher polyamines and may have

important regulatory roles in mammals [23]

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

Mn2+A and Mn2+B, respectively [9,24] However, the

volume of the active site cleft was found to be larger

for arginase II Moreover, the D232 (Od1)-Mn2+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 arginase II–BEC complex [9] The isoenzymic forms also differ in subcellular localization [8], immunologi-cal properties [8] and sensitivity to inhibition by ornithine [10], branched chain amino acids [25], fluor-ide [26] and the transition state analog S-(2-borono-ethyl)-l-cysteine [9]

In this study, the interaction of human arginase II with substrate and metal ions was examined by site-directed mutagenesis and kinetic studies Selection of target residues (Asp149, His120 and His145) was based

on the roles assigned to the equivalent residues in argi-nase I [1,24] and the crystal structure of the argiargi-nase II–BEC complex [9] The Asn149fiAsp mutation altered the substrate specificity of arginase II, yielding enzyme species with almost undetectable activity on arginine but significant activity on agmatine From the effects of replacement of His120 and His145 with aspa-ragine we conclude that Mn2+A, and not Mn2+B, as occurs in arginase I [1], is the more tightly bound ion

in arginase II

Results and Discussion

General properties of the wild-type, His120Asn, His145Asn and Asn149Asp variants of arginase II Purified wild-type, His120Asn and His145Asn variants

of arginase II were active even in the absence of added

Mn2+, although preincubation with 5 mm Mn2+ 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 Km value for l-arginine remaining essentially unaltered (Table 1) Considering His120 and His145 as metal ligands in arginase II [9], the essentially invariant Km value upon mutation of these residues agree with the currently accepted mech-anism for the arginase reaction, which considers the

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

in agreement with this, the Km value was not altered

by the full activation step Although an effect of the

mutations on substrate binding can be excluded, the

significantly lower kcat value for His120Asn indicates

that the scissible guanidino group of the substrate is

not optimally oriented with respect to the metal-bound

hydroxide in this enzyme variant The kcat and Km

values for the wild-type enzyme are comparable with

previously reported values [9]

The tryptophan fluorescence properties (kmax ¼ 338–

339 nm) and sensitivity of arginase II to thermal

inac-tivation (Fig 1) were not significantly altered by the

introduced mutations, and no differences between

the wild-type and mutant enzymes were detected by

the chromatographic procedures used for their

purifi-cation In view of these results, at least gross structural

changes can be discarded as a consequence of the

mutagenic replacements

Effects of the His120Asn and His145Asn

mutations on the affinity of metal binding

to arginase II

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 Mn2+for 10 min at 60
C

and assay in the presence of added 2 mm Mn2+, 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 Mn2+, 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 Mn2+Æsubunit)1) On the other hand, the half active wild-type and His145Asn enzymes

Table 1 Kinetic properties of the arginase activities of wild-type

and mutant variants of human arginase II Values, derived from two

separate experiments in duplicate, represent the means ± SD

Argi-nase activities were determined in 50 mm glycine ⁄ NaOH pH 9.0.

ND, Not determined, because the N149D variant expressed almost

undetectable activity on arginine.

Enzyme k cat (s -1 ) K mArg(mM) k cat ⁄ K mArg(M -1 Æs -1 )

Wild-type 249 ± 10 1.4 ± 0.1 177.9

His120Asn 131 ± 8 1.6 ± 0.1 81.9

His145Asn 238 ± 12 1.5 ± 0.1 158.6

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.

contained 1.1 ± 0.1 Mn2+Æsubunit)1 and 1.2 ± 0.1

Mn2+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

Clearly, the affinity of the arginase–manganese

inter-action was significantly altered by replacement of

His120 with asparagine This aspect was quantitatively

evaluated by following the Mn2+-dependent

reactiva-tion of metal-free species of the wild-type, His120Asn

and His145Asn variants Reactivation by free Mn2+

concentrations in the nanomolar range followed

hyper-bolic kinetics, consistent with the absence of

coopera-tivity between metal binding sites The estimated Kd

values were 1.8 ± 0.2· 10)8m for the wild-type and

His145Asn enzymes and 16.2 ± 0.5· 10)8m for the

His120Asn variant, whereas the Vmax values were

nearly equal to a half of those determined after

incu-bation of the respective enzyme variant with 5 mm

Mn2+ for 10 min at 60
C These results indicate the

existence of high and low affinity bindings of

mangan-ese ions to arginase II and provide an explanation for

the manganese stoichiometries determined here

Con-sidering the stoichiometry of 2 Mn2+Æsubunit)1derived

from EPR analysis of fully active arginase II [10], our conclusion is that a weakly bound Mn2+ 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 Mn2+, the lower affinity for that more tightly bound to the protein may explain the absence of Mn2+ 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 Mn2+ in the range of those determined here, were also reported for arginase I [28,29,30]

A binuclear motif (Mn2+A–Mn2+B) was derived from the X-ray crystal structure of fully active arginase

II complexed with BEC, a boronic acid transition state analog inhibitor of the enzyme [9] As indicated by the crystal structure, Mn2+Aand Mn2+B are, respectively, coordinated by His120 and His145, which are equival-ent to the histidines at position 101 and 126 in the sequence of arginase I [10] However, they are clearly differentiated by the consequences of their replace-ments with asparagine In fact, in contrast with argi-nase II, dialysis against EDTA results in species of the His101Asn variant of arginase I which are half active and contain 1 Mn2+Æsubunit)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 Mn2+A in argi-nase I and Mn2+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)-Mn2+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)–Mn2+B bond was also associated to the preferential release of

Mn2+B by EDTA during preparation of a substrate complex of Bacillus caldovelox arginase for crystallo-graphic analysis [32]

According to our present results, the catalytic activ-ity of the partially active species of arginase II is associated to the more tightly bound Mn2+A The increased activity resulting from the addition of the more weakly bound Mn2+B may be explained by a

Fig 2 Effect of dialysis of fully activated wild-type and mutant

species of human arginse II Fully active species were dialyzed for

4 h at 4
C against 10 m M EDTA in 10 m M Tris ⁄ HCl pH 7.5, and

then assayed for arginase 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

corres-ponding fully activated form.

lower pKa 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

coliagmatinase 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

His145Asn variants, which were practically inactive on

agmatine and markedly resistant to inhibition by the

substrate analog As an example, only about 25%

inhi-bition of the wild-type and mutant enzymes was

pro-duced by 20 mm agmatine and production of urea was practically absent using this agmatine concentration

as a potential substrate Although a more detailed ana-lysis of the effect was not performed, the inhibition by

20 mm agmatine was eliminated by saturation with

l-arginine, indicating the competitive character of the inhibition produced by the substrate analog In general agreement with our present results, agmatine was pre-viously described as a very poor alternate substrate and inhibitor for human arginase II [11]

Like the arginase activity of the wild-type, His120Asn and His145Asn variants, agmatine hydro-lysis by the Asn149Asp mutant enzyme was maximal

at pH 9–9.5 and strictly dependent on manganese ions, because metal-free species were totally inactive in the absence of added Mn2+ At the optimum pH, the

Kmfor agmatine (2.5 ± 0.2 mm) was very close to the

Km of the wild-type arginase for arginine However, the hydrolytic activity of Asn149Asp on agmatine was only about 5% of the arginase activity of the wild-type enzyme As measured by kcat⁄ Km, 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) and E coli agmatinase (d) The buffer was

50 m M glycine ⁄ NaOH pH 9.0.

cally important residues may be adopted by agmatine

in E coli agmatinase and l-arginine in B caldovelox

arginase [37], indicating that the substrate specificity

of these enzymes rely mainly in substituents at C-a

This has been, in fact, demonstrated for arginase

[38,39] and the same may be safely deduced for

agma-tinase Therefore, as an explanation for the low

cata-lytic efficiency of Asn149Asp, we conclude that the

guanidino group of agmatine is not optimally

posi-tioned and oriented for a more efficient nucleophilic

attack by a metal-bound hydroxide, most probably

due to a nonoptimal positioning of the nonguanidino

portion of the substrate molecule

Residue Asn149 is totally conserved among all the

arginases [19] and the equivalent Asn130 has been

considered as providing a hydrogen bond to the

a-carboxyl group of the substrate l-arginine in

argi-nase I [26] However, against a functional equivalence

between these residues is the observation that Asn130,

but not Asn149, interacts with the a-carboxylate

group of the transition state analog BEC in the

corresponding binary enzyme–analog complex [9]

Certainly, if an interaction between Asn149 and the

a-carboxylate group of arginine were also operative

for arginase II, both the lack of arginase activity as

well as the resistance of the Asn149Asp mutant to

inhibition by l-arginine, would be explained by

elec-trostatic repulsion between the a-carboxyl group of

the amino acid and the introduced aspartic residue at

position 149 On the other hand, as agmatine lacks

the a-carboxyl group, there would be no impediment

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

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, and enzymes and reagents for PCR were from Promega The plasmid pBluescript II K(+), bearing the gene

of human arginase II, was kindly supplied by S Cederbaum (University of California, Los Angeles) Synthetic nucleotide primers were obtained from Invitrogen and the QuickChange site-directed mutagenesis kit was from Stratagene Purified

E coliagmatinase was obtained as described previously [36] The rabbit anti-E coli agmatinase polyclonal antibody was supplied by M Salas (Universidad de Concepcio´n, Chile)

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 arginase II cDNAs were directionally cloned into the pBluescript II K(+) E coli expression vector and the enzymes were expressed in E coli strain JM109, follow-ing induction with 1 mm isopropyl thio-b-d-galactoside The bacterial cells were disrupted by sonication on ice (5· 30 s pulses) and the supernatant of a centrifugation for

20 min at 12 000 g was precipitated with ammonium sulfate (60% saturation) The pellet, recollected by centrifugation

at 12 000 g for 10 min was resuspended in 5 mm Tris⁄ HCl

pH 7.5 containing 2 mm MnCl2 and dialyzed for 6 h at

4
C against the same buffer After incubation with 5 mm MnCl2for 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

5 mm 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 MnCl2 A single protein

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

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-gene, La Jolla, CA, USA) The antisense mutagenic

oligo-nucleotide primers were: 5¢-gattgccaggctgttgtctcctcccag-3¢,

5¢-GGGGTGTGTCGATGTCA-3¢ for His120Asn, His145Asn

and Asn149Asp, respectively The corresponding sense

muta-genic oligonucleotide primers were 5¢-CTGGGAGGAGA

CAACAGCCTGGCAATC-3¢ for His120Asn, 5¢-TGGGTT

GATGCCAATGCTGACATCAAC-3¢ for His145Asn and

5¢-TGACATCGACACACCCC-3¢ for Asn149Asp

Fluorescence spectra and thermal inactivation

studies

Fluorescence measurements were made at 25
C on a

Shim-adzu RF-5301 spectrofluorimeter (Columbia, MD) The

protein concentration was 40–50 lgÆmL)1 and emission

spectra were measured with the excitation wavelength at

295 nm The slit width for both excitation and emission

was 1.5 nm, and spectra were corrected by substracting 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 75
C in a solution containing

10 mm Tris⁄ HCl pH 7.5 and 2 mm Mn2+ At several times

(up to 30 min), aliquots were removed and assayed for

residual enzymatic activity at pH 9.5, in the presence of

added 2 mm Mn2+

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 MnCl2 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

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

to a previously equilibrated buffer substrate solution Data from initial velocity studies, performed in duplicate and repeated at least twice, were fitted to the Michaelis– Menten equation, by using nonlinear regression with

USA)

To evaluate the affinity for the more tightly bound metal ion, metal-free enzymes were incubated with varied concen-trations of Mn2+in 10 mm Tris⁄ HCl pH 8.5, 50 mm KCl and 10 mm nitrilotriacetic acid as a metal ion buffer [28] After equilibration for 15 min at 37
C, arginase activities were determined in 50 mm Tris⁄ HCl pH 8.5 Free-Mn2+ concentrations were calculated using a dissociation constant

of 3.98· 10)8mand a pKa3value of 9.8 for nitrilotriacetic acid [28] Dissociation constants (Kd) and Vmaxvalues were determined from double reciprocal plots of velocity vs free metal ion concentrations

Acknowledgements

This research was supported by Grants 1030038 from FONDECYT and Grant CONICYT to support the PhD thesis of V Lo´pez

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