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

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 requirement for bivalent metal ions, especially Mn 2+ , for catalytic activity. Metal ions are thought to acti- vate a coordinated water molecule, by lowering the pK a 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 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, the His120Asn variant was totally inactive in the absence of added Mn 2+ and contained < 0.1 Mn 2+ Æsubunit )1 , whereas wild-type and His145Asn enzymes were half active and contained 1.1 ± 0.1 Mn 2+ Æsubunit )1 and 1.3 ± 0.1 Mn 2+ Æsubunit )1 , respectively. Manganese reactivation of metal- free to half active species followed hyperbolic kinetics with K d of 1.8 ± 0.2 · 10 )8 m for the wild-type and His145Asn enzymes and 16.2 ± 0.5 · 10 )8 m for the His120Asn variant. Upon mutation, the chro- matographic behavior, tryptophan fluorescence properties (k max ¼ 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, 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, 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 (Mn 2+ A –Mn 2+ 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 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 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 arginase 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 Mn 2+ A , and not Mn 2+ 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 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 in agreement with this, the K m value was not altered by the full activation step. Although an effect of the mutations on substrate binding can be excluded, the significantly lower k cat 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 k cat and K m values for the wild-type enzyme are comparable with previously reported values [9]. The tryptophan fluorescence properties (k max 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 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 )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 m Arg (mM) k cat ⁄ K m Arg (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 Asn149Asp ND ND ND 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. 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 Mn 2+ -dependent reactiva- tion of metal-free species of the wild-type, His120Asn and His145Asn variants. Reactivation by free Mn 2+ concentrations in the nanomolar range followed hyper- bolic kinetics, consistent with the absence of coopera- tivity between metal binding sites. The estimated K d values were 1.8 ± 0.2 · 10 )8 m for the wild-type and His145Asn enzymes and 16.2 ± 0.5 · 10 )8 m for the His120Asn variant, whereas the V max values were nearly equal to a half of those determined after incu- bation of the respective enzyme variant with 5 mm Mn 2+ 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 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+ in the range of those determined here, were also reported for arginase I [28,29,30]. A binuclear motif (Mn 2+ A –Mn 2+ 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, Mn 2+ A and Mn 2+ 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 Mn 2+ 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 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 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 Mn 2+ A . The increased activity resulting from the addition of the more weakly bound Mn 2+ 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 mM 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. V. Lo ´ pez et al. Interaction of arginase II with substrate and manganese ions 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 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 Mn 2+ . At the optimum pH, the K m for agmatine (2.5 ± 0.2 mm) was very close to the K m 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 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 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. coli agmatinase 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 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 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- gene, La Jolla, CA, USA). The antisense mutagenic oligo- nucleotide primers were: 5¢-gattgccaggctgttgtctcctcccag-3¢, 5¢-GTTGATGTCAGCATTGGCATCAACCCA-3¢ and 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 Mn 2+ . 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 Mn 2+ . 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 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 prism 4.0 (GraphPad Software Inc., San Diego, CA, USA). To evaluate the affinity for the more tightly bound metal ion, metal-free enzymes were incubated with varied concen- trations of Mn 2+ 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-Mn 2+ concentrations were calculated using a dissociation constant of 3.98 · 10 )8 m and a pK a3 value of 9.8 for nitrilotriacetic acid [28]. Dissociation constants (K d ) and V max values 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. References 1 Kanyo ZF, Scolnick LR, Ash DE & Christianson DW (1996) Structure of a unique binuclear manganese clus- ter in arginase. Nature 382, 554–557. 2 Christianson DW & Cox JD (1999) Catalysis by metal- activated hydroxide in zinc and manganese metallo- enzymes. Annu Rev Biochem 68, 33–57. 3 Ash DE, Cox JD & Christianson DW (2000) Arginase: a binuclear manganese metalloenzyme. Met Ions Biol Syst 37, 407–428. 4 Morris SM (2002) Regulation of enzymes of the urea cycle and arginine metabolism. Annu Rev Nutr 22, 87– 105. 5 Jenkinson CP, Grody WW & Cederbaum SD (1996) Comparative properties of arginases. Comp Biochem Physiol 114B, 107–132. Interaction of arginase II with substrate and manganese ions V. 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Insights into the interaction of human arginase II with substrate and manganese ions by site-directed mutagenesis and kinetic studies Alteration of substrate. 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 Asn14 9Asp mutations were