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: 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 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). 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In fact, whereas partial arginase activity is retained by the N130D variant of arginase I, the corresponding N149D variant of arginase. Mutational analysis of substrate recognition by human arginase type I ) agmatinase activity of the N130D variant Ricardo Alarco ´ n,