Bovine Cytosolic 5′-Nucleotidase Acts through the Formation of an Aspartate 52-Phosphoenzyme Intermediate
2001; Elsevier BV; Volume: 276; Issue: 36 Linguagem: Inglês
10.1074/jbc.m104088200
ISSN1083-351X
AutoresS. Allegrini, Andrea Scaloni, L. Ferrara, Rossana Pesi, Paolo Pinna, Francesco Sgarrella, Marcella Camici, Staffan Eriksson, Maria Grazia Tozzi,
Tópico(s)Infant Nutrition and Health
ResumoCytosolic 5′-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5′-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5′-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles. Cytosolic 5′-nucleotidase/phosphotransferase (cN-II), specific for purine monophosphates and their deoxyderivatives, acts through the formation of a phosphoenzyme intermediate. Phosphate may either be released leading to 5′-mononucleotide hydrolysis or be transferred to an appropriate nucleoside acceptor, giving rise to a mononucleotide interconversion. Chemical reagents specifically modifying aspartate and glutamate residues inhibit the enzyme, and this inhibition is partially prevented by cN-II substrates and physiological inhibitors. Peptide mapping experiments with the phosphoenzyme previously treated with tritiated borohydride allowed isolation of a radiolabeled peptide. Sequence analysis demonstrated that radioactivity was associated with a hydroxymethyl derivative that resulted from reduction of the Asp-52-phosphate intermediate. Site-directed mutagenesis experiments confirmed the essential role of Asp-52 in the catalytic machinery of the enzyme and suggested also that Asp-54 assists in the formation of the acyl phosphate species. From sequence alignments we conclude that cytosolic 5′-nucleotidase, along with other nucleotidases, belong to a large superfamily of hydrolases with different substrate specificities and functional roles. cytosolic nucleotidase l-1-tosylamido-2-phenylethyl chloromethyl ketone 2,3 bisphosphoglycerate Woodward's reagent K high performance liquid chromatography phenylthiohydantoin homoserine polymerase chain reaction matrix-assisted laser desorption ionization mass spectrometry pyrimidine nucleotidase Hydrolysis of the phosphate esterified in 3′ or 5′ of mononucleotides is catalyzed by a family of nucleotidases whose members differ in terms of substrate specificity, cellular location, regulation, distribution, and amino acid sequence. A better knowledge of the structure and catalytic mechanism of all these proteins is necessary for the understanding of their origin, evolution, and physiological significance. A classification of the enzymes specific for purine 5′-mononucleotides has been proposed by Zimmerman on the basis of cellular location and substrate specificity (1Zimmerman H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (748) Google Scholar). In vertebrates, there is a membrane-bound 5′-nucleotidase and two cytosolic 5′-nucleotidases that preferentially hydrolyses purine mononucleotides, one specific for AMP (cN-I)1 and the other specific for IMP and GMP (cN-II). Moreover, other nucleotidases specific for 5′- and 3′-deoxynucleotides located in cytosol (dNT-1) or in mitochondria (dNT-2) have been described (2Hoglund L. Reichard P. J. Biol. Chem. 1990; 265: 6589-6595Abstract Full Text PDF PubMed Google Scholar, 3Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). Furthermore, 5′-nucleotidases specific for pyrimidine mononucleotides have been described in human erythrocytes (PN-I and PN-II) (4Valentine W.N. Fink K. Paglia D.E. Harris S.R. Adams W.S. J. Clin. Invest. 1974; 54: 866-879Crossref PubMed Scopus (194) Google Scholar, 5Hirono A. Fujii H. Natori H. Kurokawa I. Miwa S. Br. J. Haematol. 1987; 65: 35-41Crossref PubMed Scopus (27) Google Scholar). Recently, PN-I has been found to be identical to p36, an α-interferon-induced protein playing a role in immune diseases (6Amici A. Emanueli M. Raffaelli N. Ruggieri S. Saccucci F. Magni G. Blood. 2000; 96: 1596-1598Crossref PubMed Google Scholar). All these proteins expressing nucleotidase activity do not show significant sequence homologies to each other (7Sala-Newby G.B. Skladanowski A.C. Newby A.C. J. Biol. Chem. 1999; 274: 17789-17793Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).cN-II has been demonstrated to act also as a phosphotransferase, which is a consequence of its reaction mechanism, proceeding through the formation of a phosphoenzyme covalent intermediate (9Worku Y. Newby A.C. Biochem. J. 1982; 205: 503-510Crossref PubMed Scopus (60) Google Scholar, 10Baiocchi C. Pesi R. Camici M. Itoh R. Tozzi M.G. Biochem. J. 1996; 317: 797-801Crossref PubMed Scopus (22) Google Scholar). No phosphotransferase activity has been reported for the other nucleotidases described so far with the exception of PN-I and PN-II (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar). cN-II is a widely expressed enzyme with a remarkable sequence conservation through evolution. Its activity is involved in the regulation of the availability of intracellular IMP (12Itoh R. Echizen H. Higuchi M. Oka J. Yamada K. Comp. Biochem. Physiol. 1992; 103B: 153-159Google Scholar). Because IMP is the precursor of all purine nucleotides, which are not only nucleic acid building blocks but also energy transducers, intracellular and extra cellular signals, and metabolic regulators, the regulation of its hydrolysis is of paramount importance for a number of cell functions. Recently, an increased activity of cN-II has been associated to a developmental neurological disorder and to Lesch-Nyhan syndrome (13Page T., Yu, A. Fontanesi J. Nyhan W.L. Proc. Natl. Acad. Sci. 1997; 94: 11601-11606Crossref PubMed Scopus (74) Google Scholar,14Pesi R. Micheli V. Jacomelli G. Peruzzi L. Camici M. Garcia-Gil M. Allegrini S. Tozzi M.G. Neuroreport. 2000; 11: 1827-1831Crossref PubMed Scopus (53) Google Scholar).In this work we provide evidence that cN-II becomes phosphorylated during the catalytic cycle on the first aspartate (Asp-52) occurring in a DMDYT motif conserved among the different species. This consensus sequence is also shared with other members of the 5′-nucleotidases family. In addition, a DXDX(T/V) motif has been described as being involved in the catalytic mechanism of a number of other phosphatases/phosphotransferases (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), suggesting a common mechanism of action for all these enzymes that do not present any significant sequence homology with cN-II.DISCUSSIONThe results reported in this work demonstrate that cN-II is inhibited by WRK in a dose-dependent manner, suggesting that this reagent modifies carboxylate residues important for proper enzymatic functioning. WRK is well established in terms of its ability to modify aspartates or glutamates (27Keresztessy Z. Kiss L. Hughes M.A. Arch. Biochem. Biophys. 1994; 314: 142-152Crossref PubMed Scopus (36) Google Scholar, 28Kommisarov A.A. Romanova D.V. Debabov V.G. J. Biol. Chem. 1995; 270: 10050-10055Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The selection of appropriate reaction conditions (pH 6.0 and very short inactivation times) ensured us an increased selectivity of WRK for acid residues and ruled out a possible reaction of this compound with other nucleophile amino acids (29Maralihalli G.B. Bhagwat A.S. J. Protein Chem. 1993; 12: 451-457Crossref PubMed Scopus (10) Google Scholar, 30Bustos P. Gajardo M.I. Gomez C. Goldie H. Cardemil E. Jabalquinto A.M. J. Protein Chem. 1996; 15: 467-472Crossref PubMed Scopus (15) Google Scholar, 31Llamas K. Owens M. Blakeley R.L. Zerner B. J. Am. Chem. Soc. 1986; 108: 5543-5548Crossref Scopus (27) Google Scholar). The partial protection exerted by substrates and inhibitors of the enzyme is strongly indicative that some of the modified residues are located in the active site. It was previously observed that the allosteric inhibitor phosphate causes an increase in the Km for IMP, suggesting that the cN-II active site can result less accessibly in the presence of this anion. On the contrary, has been reported that the allosteric activator ATP, in the presence of Mg2+ causes a moderate decrease in theKm for the substrate and an increase in the catalytic rate (32Pesi R. Turriani M. Allegrini S. Scolozzi C. Camici M. Ipata P.L. Tozzi M.G. Arch. Biochem. Biophys. 1994; 312: 75-80Crossref PubMed Scopus (76) Google Scholar). We find here that phosphate is effective in protection against WRK inactivation, while ATP-Mg2+, which increase the accessibility of the active site, do not exert any protective effect. On the contrary, ATP in the absence of Mg2+ indeed afforded a protection similar to that observed for IMP. ATP is also stabilizing the enzyme during storage and against thermal inactivation (12Itoh R. Echizen H. Higuchi M. Oka J. Yamada K. Comp. Biochem. Physiol. 1992; 103B: 153-159Google Scholar). These observations indicate that, in the absence of Mg2+, ATP can determine a protective effect possibly by an interaction with the active site.In a previous paper we demonstrated that cN-II forms a phosphoenzyme intermediate during catalysis, which was extremely labile (10Baiocchi C. Pesi R. Camici M. Itoh R. Tozzi M.G. Biochem. J. 1996; 317: 797-801Crossref PubMed Scopus (22) Google Scholar). On the basis of this observation and the enzyme sensitivity toward treatment with WRK, we deduced that an aspartate or glutamate residue was the phosphate acceptor. The identification of this amino acid has been obtained after reduction of the acyl phosphate intermediate with radiolabeled borohydride, and separation of its tryptic digest, radioactivity, and molecular mass measurements. Sequence analysis performed on the isolated peptide demonstrated that Asp-52 was the phosphorylated residue. Site-directed mutagenesis experiments confirmed the fundamental role of this residue for the enzyme activity; in fact, even a conservative substitution in this position totally abolished enzyme activity and prevented the formation of the phosphorylated enzyme. In addition, mutants on Asp-54 were inactive and not able to generate this intermediate. On this basis, our results demonstrate that cN-II belongs to the large class of phosphohydrolases described by Collet et al. (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) because it shows close to the N terminus a DXDX(T/V) motif in which the first Asp is phosphorylated during catalysis. Although not so structurally similar to cN-II to be related by a simple sequence analysis investigation, most of these enzymes present a phosphotransferase-phosphatase activity, a common catalytic dependence from Mg2+ and the occurrence of a phosphointermediate. As shown in Fig. 6, all the proteins listed have the common motif flanked by almost conserved hydrophobic amino acids. A few months ago, the three-dimensional structure of the first member of this family, phosphoserine phosphatase fromMethanococcus jannaschii, had been solved by x-ray crystallography (33Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This enzyme presents a fold consisting of separate α/β and four-helix-bundle domains. A careful structural analysis revealed the simultaneous occurrence of four acid residues into its active site. Asp-11 is important in playing the role of phosphate acceptor; similarly, Asp-13, Asp-167, and Glu-20 seem to be essential for Mg2+ coordination. Site-directed mutagenesis experiments revealed the importance of these residues in catalysis. The data reported in this paper on the stoichiometry of the cN-II inactivation by WRK demonstrate that, also in this case, four acid residues are modified with the same efficiency. Inactivation is protected by enzyme substrates and inhibitors, suggesting that modified residues are located into the active site. In addition, both Asp residues occurring in the DXDX(T/V) motif of cN-II (Asp-52 and Asp-54), similar to that of phosphoserine phosphatase (Asp-11 and Asp-13), are essential for catalysis. Therefore, these data are in strict analogy with that determined for phosphoserine phosphatase and suggest that also in cN-II the occurrence of different acid residues in the active site is important for Mg2+ coordination and catalytic efficiency.Furthermore, sequence comparison of cN-II with other nucleotidases demonstrates that also pyrimidine nucleotidase purified from human erythrocytes (PN-I) and 5′-3′-deoxynucleotidase located in the cytoplasm (dNT-1) and in its mitochondrial counterpart (dNT-2), contain a DXDX(T/V) motif. Also human cN-I shows a similar sequence even though located close to the C terminus. Pyrimidine nucleotidase PN-I has been shown to have a phosphotransferase activity (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar), which implicates a reaction mechanism proceeding through a phosphorylated enzyme intermediate. In the case of dNT-1 and dNT-2, the reaction mechanism has not been described so far. The two enzymes present a high degree of identity in their structure and molecular and functional characteristics, suggesting that they act through the same mechanism. dNT-1 has been recently cloned and expressed in Escherichia coli and mammalian cells (8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and was found remarkably similar to PN-II described in human erythrocytes concluding that PN-II, which is a phosphatase/phosphotransferase (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar), actually belongs to the class of the dNT's (8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Furthermore, substrate specificity of dNT-1 is remarkably similar to that displayed by the phosphotransferase with hydrolase activity acting on deoxynucleotides described by Tesoriere etal. (34Tesoriere G. Vento R. Tesoriere L. Giuliano M. Biochim. Biophys. Acta. 1984; 786: 231-244Crossref PubMed Scopus (6) Google Scholar). Human cN-I has been recently cloned (35Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), but its reaction mechanism has not been described so far. However its functional similarities with cN-II (such as the complete Mg2+ dependence) suggest that it might have a similar reaction mechanism. Therefore, on the basis of these observations and the sequence alignment reported in Fig. 6 we can presumably assume that catalysis of all soluble nucleotidases proceed through a similar reaction mechanism involving the formation of a phosphorylated intermediate. Therefore, we conclude that cN-II is a first example of a group of eukaryotic cytosolic nucleotidases presenting a common catalytic machinery and conserved active site residues that resemble those occurring in other enzymes belonging to the superfamily of bacterial, eukaryotic and archeal phosphohydrolases (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 26Aravidin L. Michael Y. Koonin G. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 33Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Hydrolysis of the phosphate esterified in 3′ or 5′ of mononucleotides is catalyzed by a family of nucleotidases whose members differ in terms of substrate specificity, cellular location, regulation, distribution, and amino acid sequence. A better knowledge of the structure and catalytic mechanism of all these proteins is necessary for the understanding of their origin, evolution, and physiological significance. A classification of the enzymes specific for purine 5′-mononucleotides has been proposed by Zimmerman on the basis of cellular location and substrate specificity (1Zimmerman H. Biochem. J. 1992; 285: 345-365Crossref PubMed Scopus (748) Google Scholar). In vertebrates, there is a membrane-bound 5′-nucleotidase and two cytosolic 5′-nucleotidases that preferentially hydrolyses purine mononucleotides, one specific for AMP (cN-I)1 and the other specific for IMP and GMP (cN-II). Moreover, other nucleotidases specific for 5′- and 3′-deoxynucleotides located in cytosol (dNT-1) or in mitochondria (dNT-2) have been described (2Hoglund L. Reichard P. J. Biol. Chem. 1990; 265: 6589-6595Abstract Full Text PDF PubMed Google Scholar, 3Rampazzo C. Gallinaro L. Milanesi E. Frigimelica E. Reichard P. Bianchi V. Proc. Natl. Acad. Sci. 2000; 97: 8239-8244Crossref PubMed Scopus (84) Google Scholar). Furthermore, 5′-nucleotidases specific for pyrimidine mononucleotides have been described in human erythrocytes (PN-I and PN-II) (4Valentine W.N. Fink K. Paglia D.E. Harris S.R. Adams W.S. J. Clin. Invest. 1974; 54: 866-879Crossref PubMed Scopus (194) Google Scholar, 5Hirono A. Fujii H. Natori H. Kurokawa I. Miwa S. Br. J. Haematol. 1987; 65: 35-41Crossref PubMed Scopus (27) Google Scholar). Recently, PN-I has been found to be identical to p36, an α-interferon-induced protein playing a role in immune diseases (6Amici A. Emanueli M. Raffaelli N. Ruggieri S. Saccucci F. Magni G. Blood. 2000; 96: 1596-1598Crossref PubMed Google Scholar). All these proteins expressing nucleotidase activity do not show significant sequence homologies to each other (7Sala-Newby G.B. Skladanowski A.C. Newby A.C. J. Biol. Chem. 1999; 274: 17789-17793Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). cN-II has been demonstrated to act also as a phosphotransferase, which is a consequence of its reaction mechanism, proceeding through the formation of a phosphoenzyme covalent intermediate (9Worku Y. Newby A.C. Biochem. J. 1982; 205: 503-510Crossref PubMed Scopus (60) Google Scholar, 10Baiocchi C. Pesi R. Camici M. Itoh R. Tozzi M.G. Biochem. J. 1996; 317: 797-801Crossref PubMed Scopus (22) Google Scholar). No phosphotransferase activity has been reported for the other nucleotidases described so far with the exception of PN-I and PN-II (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar). cN-II is a widely expressed enzyme with a remarkable sequence conservation through evolution. Its activity is involved in the regulation of the availability of intracellular IMP (12Itoh R. Echizen H. Higuchi M. Oka J. Yamada K. Comp. Biochem. Physiol. 1992; 103B: 153-159Google Scholar). Because IMP is the precursor of all purine nucleotides, which are not only nucleic acid building blocks but also energy transducers, intracellular and extra cellular signals, and metabolic regulators, the regulation of its hydrolysis is of paramount importance for a number of cell functions. Recently, an increased activity of cN-II has been associated to a developmental neurological disorder and to Lesch-Nyhan syndrome (13Page T., Yu, A. Fontanesi J. Nyhan W.L. Proc. Natl. Acad. Sci. 1997; 94: 11601-11606Crossref PubMed Scopus (74) Google Scholar,14Pesi R. Micheli V. Jacomelli G. Peruzzi L. Camici M. Garcia-Gil M. Allegrini S. Tozzi M.G. Neuroreport. 2000; 11: 1827-1831Crossref PubMed Scopus (53) Google Scholar). In this work we provide evidence that cN-II becomes phosphorylated during the catalytic cycle on the first aspartate (Asp-52) occurring in a DMDYT motif conserved among the different species. This consensus sequence is also shared with other members of the 5′-nucleotidases family. In addition, a DXDX(T/V) motif has been described as being involved in the catalytic mechanism of a number of other phosphatases/phosphotransferases (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), suggesting a common mechanism of action for all these enzymes that do not present any significant sequence homology with cN-II. DISCUSSIONThe results reported in this work demonstrate that cN-II is inhibited by WRK in a dose-dependent manner, suggesting that this reagent modifies carboxylate residues important for proper enzymatic functioning. WRK is well established in terms of its ability to modify aspartates or glutamates (27Keresztessy Z. Kiss L. Hughes M.A. Arch. Biochem. Biophys. 1994; 314: 142-152Crossref PubMed Scopus (36) Google Scholar, 28Kommisarov A.A. Romanova D.V. Debabov V.G. J. Biol. Chem. 1995; 270: 10050-10055Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The selection of appropriate reaction conditions (pH 6.0 and very short inactivation times) ensured us an increased selectivity of WRK for acid residues and ruled out a possible reaction of this compound with other nucleophile amino acids (29Maralihalli G.B. Bhagwat A.S. J. Protein Chem. 1993; 12: 451-457Crossref PubMed Scopus (10) Google Scholar, 30Bustos P. Gajardo M.I. Gomez C. Goldie H. Cardemil E. Jabalquinto A.M. J. Protein Chem. 1996; 15: 467-472Crossref PubMed Scopus (15) Google Scholar, 31Llamas K. Owens M. Blakeley R.L. Zerner B. J. Am. Chem. Soc. 1986; 108: 5543-5548Crossref Scopus (27) Google Scholar). The partial protection exerted by substrates and inhibitors of the enzyme is strongly indicative that some of the modified residues are located in the active site. It was previously observed that the allosteric inhibitor phosphate causes an increase in the Km for IMP, suggesting that the cN-II active site can result less accessibly in the presence of this anion. On the contrary, has been reported that the allosteric activator ATP, in the presence of Mg2+ causes a moderate decrease in theKm for the substrate and an increase in the catalytic rate (32Pesi R. Turriani M. Allegrini S. Scolozzi C. Camici M. Ipata P.L. Tozzi M.G. Arch. Biochem. Biophys. 1994; 312: 75-80Crossref PubMed Scopus (76) Google Scholar). We find here that phosphate is effective in protection against WRK inactivation, while ATP-Mg2+, which increase the accessibility of the active site, do not exert any protective effect. On the contrary, ATP in the absence of Mg2+ indeed afforded a protection similar to that observed for IMP. ATP is also stabilizing the enzyme during storage and against thermal inactivation (12Itoh R. Echizen H. Higuchi M. Oka J. Yamada K. Comp. Biochem. Physiol. 1992; 103B: 153-159Google Scholar). These observations indicate that, in the absence of Mg2+, ATP can determine a protective effect possibly by an interaction with the active site.In a previous paper we demonstrated that cN-II forms a phosphoenzyme intermediate during catalysis, which was extremely labile (10Baiocchi C. Pesi R. Camici M. Itoh R. Tozzi M.G. Biochem. J. 1996; 317: 797-801Crossref PubMed Scopus (22) Google Scholar). On the basis of this observation and the enzyme sensitivity toward treatment with WRK, we deduced that an aspartate or glutamate residue was the phosphate acceptor. The identification of this amino acid has been obtained after reduction of the acyl phosphate intermediate with radiolabeled borohydride, and separation of its tryptic digest, radioactivity, and molecular mass measurements. Sequence analysis performed on the isolated peptide demonstrated that Asp-52 was the phosphorylated residue. Site-directed mutagenesis experiments confirmed the fundamental role of this residue for the enzyme activity; in fact, even a conservative substitution in this position totally abolished enzyme activity and prevented the formation of the phosphorylated enzyme. In addition, mutants on Asp-54 were inactive and not able to generate this intermediate. On this basis, our results demonstrate that cN-II belongs to the large class of phosphohydrolases described by Collet et al. (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) because it shows close to the N terminus a DXDX(T/V) motif in which the first Asp is phosphorylated during catalysis. Although not so structurally similar to cN-II to be related by a simple sequence analysis investigation, most of these enzymes present a phosphotransferase-phosphatase activity, a common catalytic dependence from Mg2+ and the occurrence of a phosphointermediate. As shown in Fig. 6, all the proteins listed have the common motif flanked by almost conserved hydrophobic amino acids. A few months ago, the three-dimensional structure of the first member of this family, phosphoserine phosphatase fromMethanococcus jannaschii, had been solved by x-ray crystallography (33Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This enzyme presents a fold consisting of separate α/β and four-helix-bundle domains. A careful structural analysis revealed the simultaneous occurrence of four acid residues into its active site. Asp-11 is important in playing the role of phosphate acceptor; similarly, Asp-13, Asp-167, and Glu-20 seem to be essential for Mg2+ coordination. Site-directed mutagenesis experiments revealed the importance of these residues in catalysis. The data reported in this paper on the stoichiometry of the cN-II inactivation by WRK demonstrate that, also in this case, four acid residues are modified with the same efficiency. Inactivation is protected by enzyme substrates and inhibitors, suggesting that modified residues are located into the active site. In addition, both Asp residues occurring in the DXDX(T/V) motif of cN-II (Asp-52 and Asp-54), similar to that of phosphoserine phosphatase (Asp-11 and Asp-13), are essential for catalysis. Therefore, these data are in strict analogy with that determined for phosphoserine phosphatase and suggest that also in cN-II the occurrence of different acid residues in the active site is important for Mg2+ coordination and catalytic efficiency.Furthermore, sequence comparison of cN-II with other nucleotidases demonstrates that also pyrimidine nucleotidase purified from human erythrocytes (PN-I) and 5′-3′-deoxynucleotidase located in the cytoplasm (dNT-1) and in its mitochondrial counterpart (dNT-2), contain a DXDX(T/V) motif. Also human cN-I shows a similar sequence even though located close to the C terminus. Pyrimidine nucleotidase PN-I has been shown to have a phosphotransferase activity (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar), which implicates a reaction mechanism proceeding through a phosphorylated enzyme intermediate. In the case of dNT-1 and dNT-2, the reaction mechanism has not been described so far. The two enzymes present a high degree of identity in their structure and molecular and functional characteristics, suggesting that they act through the same mechanism. dNT-1 has been recently cloned and expressed in Escherichia coli and mammalian cells (8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and was found remarkably similar to PN-II described in human erythrocytes concluding that PN-II, which is a phosphatase/phosphotransferase (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar), actually belongs to the class of the dNT's (8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Furthermore, substrate specificity of dNT-1 is remarkably similar to that displayed by the phosphotransferase with hydrolase activity acting on deoxynucleotides described by Tesoriere etal. (34Tesoriere G. Vento R. Tesoriere L. Giuliano M. Biochim. Biophys. Acta. 1984; 786: 231-244Crossref PubMed Scopus (6) Google Scholar). Human cN-I has been recently cloned (35Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), but its reaction mechanism has not been described so far. However its functional similarities with cN-II (such as the complete Mg2+ dependence) suggest that it might have a similar reaction mechanism. Therefore, on the basis of these observations and the sequence alignment reported in Fig. 6 we can presumably assume that catalysis of all soluble nucleotidases proceed through a similar reaction mechanism involving the formation of a phosphorylated intermediate. Therefore, we conclude that cN-II is a first example of a group of eukaryotic cytosolic nucleotidases presenting a common catalytic machinery and conserved active site residues that resemble those occurring in other enzymes belonging to the superfamily of bacterial, eukaryotic and archeal phosphohydrolases (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 26Aravidin L. Michael Y. Koonin G. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 33Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). The results reported in this work demonstrate that cN-II is inhibited by WRK in a dose-dependent manner, suggesting that this reagent modifies carboxylate residues important for proper enzymatic functioning. WRK is well established in terms of its ability to modify aspartates or glutamates (27Keresztessy Z. Kiss L. Hughes M.A. Arch. Biochem. Biophys. 1994; 314: 142-152Crossref PubMed Scopus (36) Google Scholar, 28Kommisarov A.A. Romanova D.V. Debabov V.G. J. Biol. Chem. 1995; 270: 10050-10055Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The selection of appropriate reaction conditions (pH 6.0 and very short inactivation times) ensured us an increased selectivity of WRK for acid residues and ruled out a possible reaction of this compound with other nucleophile amino acids (29Maralihalli G.B. Bhagwat A.S. J. Protein Chem. 1993; 12: 451-457Crossref PubMed Scopus (10) Google Scholar, 30Bustos P. Gajardo M.I. Gomez C. Goldie H. Cardemil E. Jabalquinto A.M. J. Protein Chem. 1996; 15: 467-472Crossref PubMed Scopus (15) Google Scholar, 31Llamas K. Owens M. Blakeley R.L. Zerner B. J. Am. Chem. Soc. 1986; 108: 5543-5548Crossref Scopus (27) Google Scholar). The partial protection exerted by substrates and inhibitors of the enzyme is strongly indicative that some of the modified residues are located in the active site. It was previously observed that the allosteric inhibitor phosphate causes an increase in the Km for IMP, suggesting that the cN-II active site can result less accessibly in the presence of this anion. On the contrary, has been reported that the allosteric activator ATP, in the presence of Mg2+ causes a moderate decrease in theKm for the substrate and an increase in the catalytic rate (32Pesi R. Turriani M. Allegrini S. Scolozzi C. Camici M. Ipata P.L. Tozzi M.G. Arch. Biochem. Biophys. 1994; 312: 75-80Crossref PubMed Scopus (76) Google Scholar). We find here that phosphate is effective in protection against WRK inactivation, while ATP-Mg2+, which increase the accessibility of the active site, do not exert any protective effect. On the contrary, ATP in the absence of Mg2+ indeed afforded a protection similar to that observed for IMP. ATP is also stabilizing the enzyme during storage and against thermal inactivation (12Itoh R. Echizen H. Higuchi M. Oka J. Yamada K. Comp. Biochem. Physiol. 1992; 103B: 153-159Google Scholar). These observations indicate that, in the absence of Mg2+, ATP can determine a protective effect possibly by an interaction with the active site. In a previous paper we demonstrated that cN-II forms a phosphoenzyme intermediate during catalysis, which was extremely labile (10Baiocchi C. Pesi R. Camici M. Itoh R. Tozzi M.G. Biochem. J. 1996; 317: 797-801Crossref PubMed Scopus (22) Google Scholar). On the basis of this observation and the enzyme sensitivity toward treatment with WRK, we deduced that an aspartate or glutamate residue was the phosphate acceptor. The identification of this amino acid has been obtained after reduction of the acyl phosphate intermediate with radiolabeled borohydride, and separation of its tryptic digest, radioactivity, and molecular mass measurements. Sequence analysis performed on the isolated peptide demonstrated that Asp-52 was the phosphorylated residue. Site-directed mutagenesis experiments confirmed the fundamental role of this residue for the enzyme activity; in fact, even a conservative substitution in this position totally abolished enzyme activity and prevented the formation of the phosphorylated enzyme. In addition, mutants on Asp-54 were inactive and not able to generate this intermediate. On this basis, our results demonstrate that cN-II belongs to the large class of phosphohydrolases described by Collet et al. (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar) because it shows close to the N terminus a DXDX(T/V) motif in which the first Asp is phosphorylated during catalysis. Although not so structurally similar to cN-II to be related by a simple sequence analysis investigation, most of these enzymes present a phosphotransferase-phosphatase activity, a common catalytic dependence from Mg2+ and the occurrence of a phosphointermediate. As shown in Fig. 6, all the proteins listed have the common motif flanked by almost conserved hydrophobic amino acids. A few months ago, the three-dimensional structure of the first member of this family, phosphoserine phosphatase fromMethanococcus jannaschii, had been solved by x-ray crystallography (33Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). This enzyme presents a fold consisting of separate α/β and four-helix-bundle domains. A careful structural analysis revealed the simultaneous occurrence of four acid residues into its active site. Asp-11 is important in playing the role of phosphate acceptor; similarly, Asp-13, Asp-167, and Glu-20 seem to be essential for Mg2+ coordination. Site-directed mutagenesis experiments revealed the importance of these residues in catalysis. The data reported in this paper on the stoichiometry of the cN-II inactivation by WRK demonstrate that, also in this case, four acid residues are modified with the same efficiency. Inactivation is protected by enzyme substrates and inhibitors, suggesting that modified residues are located into the active site. In addition, both Asp residues occurring in the DXDX(T/V) motif of cN-II (Asp-52 and Asp-54), similar to that of phosphoserine phosphatase (Asp-11 and Asp-13), are essential for catalysis. Therefore, these data are in strict analogy with that determined for phosphoserine phosphatase and suggest that also in cN-II the occurrence of different acid residues in the active site is important for Mg2+ coordination and catalytic efficiency. Furthermore, sequence comparison of cN-II with other nucleotidases demonstrates that also pyrimidine nucleotidase purified from human erythrocytes (PN-I) and 5′-3′-deoxynucleotidase located in the cytoplasm (dNT-1) and in its mitochondrial counterpart (dNT-2), contain a DXDX(T/V) motif. Also human cN-I shows a similar sequence even though located close to the C terminus. Pyrimidine nucleotidase PN-I has been shown to have a phosphotransferase activity (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar), which implicates a reaction mechanism proceeding through a phosphorylated enzyme intermediate. In the case of dNT-1 and dNT-2, the reaction mechanism has not been described so far. The two enzymes present a high degree of identity in their structure and molecular and functional characteristics, suggesting that they act through the same mechanism. dNT-1 has been recently cloned and expressed in Escherichia coli and mammalian cells (8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and was found remarkably similar to PN-II described in human erythrocytes concluding that PN-II, which is a phosphatase/phosphotransferase (11Amici A. Emanuelli M. Magni G. Raffaelli N. Ruggeri S. FEBS Lett. 1997; 419: 263-267Crossref PubMed Scopus (50) Google Scholar), actually belongs to the class of the dNT's (8Rampazzo C. Johansson M. Gallinaro L. Ferraro P. Hellman U. Karlsson A. Reichard P. Bianchi V. J. Biol. Chem. 2000; 275: 5409-5415Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Furthermore, substrate specificity of dNT-1 is remarkably similar to that displayed by the phosphotransferase with hydrolase activity acting on deoxynucleotides described by Tesoriere etal. (34Tesoriere G. Vento R. Tesoriere L. Giuliano M. Biochim. Biophys. Acta. 1984; 786: 231-244Crossref PubMed Scopus (6) Google Scholar). Human cN-I has been recently cloned (35Hunsucker S.A. Spychala J. Mitchell B.S. J. Biol. Chem. 2001; 276: 10498-10504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), but its reaction mechanism has not been described so far. However its functional similarities with cN-II (such as the complete Mg2+ dependence) suggest that it might have a similar reaction mechanism. Therefore, on the basis of these observations and the sequence alignment reported in Fig. 6 we can presumably assume that catalysis of all soluble nucleotidases proceed through a similar reaction mechanism involving the formation of a phosphorylated intermediate. Therefore, we conclude that cN-II is a first example of a group of eukaryotic cytosolic nucleotidases presenting a common catalytic machinery and conserved active site residues that resemble those occurring in other enzymes belonging to the superfamily of bacterial, eukaryotic and archeal phosphohydrolases (15Collet J.F. Stroobant V. Pirard M. Delpierre G. Van Schaftingen E. J. Biol. Chem. 1998; 273: 14107-14112Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 26Aravidin L. Michael Y. Koonin G. Koonin E.V. Trends Biochem. Sci. 1998; 23: 127-129Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, 33Wang W. Kim R. Jancarik J. Yokota H. Kim S.H. Structure. 2001; 9: 65-71Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). We thank Dr. J. F. Collet for the helpful suggestions and criticism.
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