Mechanistic studies on bovine cytosolic 5¢-nucleotidase II, an enzyme belonging to the HAD superfamily Simone Allegrini 1, *, Andrea Scaloni 2, *, Maria Giovanna Careddu 1 , Giovanna Cuccu 3 , Chiara D’Ambrosio 2 , Rossana Pesi 3, *, Marcella Camici 3 , Lino Ferrara 2 and Maria Grazia Tozzi 3 1 Dipartimento di Scienze del Farmaco, Universita ` di Sassari, Italy; 2 Proteomics and Mass Spectrometry Laboratory, ISPAAM, National Research Council, Naples, Italy; 3 Dipartimento di Fisiologia e Biochimica, Universita ` di Pisa, Italy Cytosolic 5¢-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through t he formation o f an enzyme–phosphate intermediate. Sequence alignment with members of the P-type ATPases/L-2-halo- acid dehalogenase superfamily identified three highly con- served motifs in cN-II and other cytosolic nucleotidases. Mutagenesis studies at specific amino acids occurring in cN-II conserved motifs were performed. The modification of the m easured k inetic parameter s, c aused by conservative a nd nonconservative substitutions, suggested that motif I is involved in the formation and stabilization of t he covalent enzyme–phosphate intermediate. Similarly, T249 in motif II as well as K292 in motif III also contribute to stabilize the phospho–enzyme adduct. Finally, D351 and D356 in motif III coordinate magnesium ion, which is required for cata- lysis. These findings were consistent with data already determined for P -type ATPases, haloacid dehalogenases and phosphotransferases, thus suggesting that cN-II and other mammalian 5¢-nucleotidases are characterized by a 3D arrangement related to the 2-haloacid dehalogenase super- fold. Structural determinants involved in differential regu- lation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated b y proteolysis were ascertained by combined biochemical and mass spectrometric investigations. These experiments indicated that the C-terminal r egion of cN-II contains a cysteine prone to form a disulfide bond, thereby inactivating the enzyme. Proteolysis events that generate the observed cN-II forms, eliminating this C-terminal portion, may prevent loss of enzymic activity and can be regarded a s regulatory pheno- mena. Keywords: catalytic residues ; HAD; nucleotidase; regulation; site-directed mutagenesis. Mammalian 5¢-nucleotidases (eN, cN-Ia, cN-Ib, cN-II, cN-III, cdN and mdN) make up a family of proteins with different subcellular locations and remarkably low sequence similarities [1]. Besides ectosolic 5¢-nucleotidase, one mito- chondrial and five cytosolic enzymes have been described to date. According to its substrate specificity and tissue distribution, each protein seems to play a specific role within the cell. In fact, cN-Is, which is highly expressed in skeletal muscle, heart and testis, is specific for AMP and seems to be involved in adenosine production during hypoxia or ischemia, because it mediates the cell response to low energy charges [2]. On the other hand, cN-II is more specific for i nosine monophosphate (IMP) and GMP, and i s a ubiquitous enzyme involved in the regulation of intracellular IMP and GMP concentrations [3]. Furthermore, cN-III, which is expressed in red blood cells and is specific for pyrimidines, seems to participate in RNA degradation during erythrocyte maturation [4]. Likewise, cytosolic and mitochondrial deoxynucleotidases (cdN and mdN) regulate nucleotide pools in their respective compartments [1]. cN-II was the first member of the cytosolic 5¢-nucleotid- ases whose r eaction mechanism was elucidated [5]. During catalysis, this enzyme was shown to become phosphorylated on the first aspartate of its DMDYT sequence. A similar motif DXDX(T/V) (motif I) is present in all members of the HAD superfamily, where the nucleophilic attack of this aspartate is essential for the catalytic machinery [6–8]. P-type ATPase/phosphotransferase members of the HAD superfamily share a similar structural fold and a co mmon reaction mechanism, which requires the formation o f a covalent enzyme–phosphate inte rmediate [8]. Furthermore, crystallographic and site-directed mutagenesis studies on these p roteins have demonst rated that a series of other common amino acids always occur in their active site [7–9], thus confirming the presence of two additio nal sequence motifs common to all members of the HAD family [8,9]. The first (motif II) is characterized by a threonine/serine residue included in a hydrophobic region; the second (motif III) presents a conserved lysine and a pair of aspartic acid Correspondence to S. Allegrini, Universita ` di Sassari, Dipartimento di Scienze del Farmaco, via Muroni 23/A, 07100 Sassari, Italy. Fax: +39 079 228708, Tel .: +39 079 228715, E-mail: enomis@uniss.it Abbreviations: BPG, 2,3-biphosphoglycerate; CAM, carboxyamido- methylated; cdN, cytosolic deoxynucleotidase; cN, cytosolic nucleo- tidase; eN, ectosolic nucleotidase; HAD, L-2-haloacid dehalogenase; IMP, inosine monophosphate; mdN, mitochondrial deoxynucleoti- dase; PSP, phosphoserine phosphatase. *Note: Th ese authors c ontributed e qu ally to t he w ork p resent ed in this article. (Received 3 August 2004, revised 11 October 2004, accepted 25 October 2004) Eur. J. Biochem. 271, 4881–4891 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04457.x residues. Very recently, the resolution of the crystal structure of mdN, a dimeric mitochondrial nucleotidase specific for deoxynucleotides, has been reported, proving that this enzyme is the first example of a 5¢-nucleotidase belonging to the HAD superfamily [7]. On this basis, a large number of proteins differing in catalytic activity against various substrates, polypeptide length (from 200 to 1400 amino acids), domain arrangement, oligomerization and conform- ational change following ligand binding, have been related to the H AD/P-type ATPases/phosphotransferases s uper- fold [10]. However, no structural data on cN-II are currently available. Unlike other 5¢-nucleotidases, cN-II activity is modula- ted by various ligands; it is activated by ADP, ATP, 2,3-biphosphoglycerate (BPG) and decavanadate, and is inhibited by phosphate. On the basis of these regulatory properties, its physiological role has been hypothesized as being associated with the hydrolysis o f excess IMP that has been newly synthesized or salvaged in the presence of a high- energy charge [11]. The enzyme generates inosine, which, in turn, can leave the cell and/or be converted into hypoxan- thine and uric acid. However, when I MP accumulates as a consequence of ATP hydrolysis, cN-II becomes virtually inactive, allowing the accumulation of the monophosphate and p reventing the loss of precious purine molecules [3,11–14]. T wo enzyme forms of bovine cN-II have been reported, which can be distinguished in terms of electroph- oretic, chromatographic a nd regu latory characteristics [ 13]. The physiological relevance of t his observation remains obscure, together with the nature (either genetic or regula- tive) of the mechanisms generating these species. Moreover, cN-II presents both phosphatase and phosphotransferase activities. Even though the physiological re levance of the phosphotransferase activity is not clear, the enzyme has been demo nstrated as being responsible for the phosphory- lation of nucleoside analogs in use as a ntineoplastic and antiviral drugs [15,16]. Furthermore, c N-II seems to b e responsible for t he resistance to several purine derivative drugs [17,18]. Therefore, it would seem that cN-II plays a fundamental role in the effectiveness of several purine drugs and its activity may be predictive of patient survival in acute myeloid leukaemia [19]. Finally, cN-II overactivity has been demonstrated in Lesch–Nyhan syndrome, which might be associated with neurological symptoms related to this disease [20–22]. For these reasons, biochemical studies, aimed at com- pletely elucidating the cN-II structure with re spect to its functional and regulatory properties, are particularly important. In fact, these investigations will be fundamental for the design of nucleoside derivatives that could interfere with enzyme function and stability, thus playing a role both in the therapy of malignancies and neurological disorders caused by purine dismetabolisms. In this article, we report the kinetic characte rization of a series of cN-II mutants, designed on the basis o f sequence alignment with P-type ATPases, haloacid dehalogenases and phosphotransferases. Our results indicate that cN-II presents an active site strongly resembling those present in other members of the HAD superfamily. Furthermore, we investigated t he struc- tural determinants involved in the regulation of cN-II activity in the presence of ligands or redox reagents by using a combined biochemical and p roteomic approach. Experimental procedures Materials Talon metal affinity resin was from Clontech Laboratories (Palo Alto, CA, USA). [8- 14 C]Inosine was purchased from Sigma Chemical Co. (St Louis, MO, USA). Thrombin was from Amersham Pharma cia Biotech (Uppsala, Sweden). Poly(vinylidene d ifluoride) (PVDF) membrane was pur- chased from Millipore Co. (Billerica, MA, USA). G oose anticytosolic 5¢-nucleotidase (from pig lung) IgG and rabbit anti-goose IgG serum were kind gifts from R. I toh ( Tokyo Kasei Gakuin University, Tokyo, Japan). All other chem- icals were reagent grade. All s olvents were HPLC grade. Sequence alignment Iterated sequence comparisons and position-specific iterated PSI-BLAST search results, starting from P-type ATPases and H AD, were used as starting multiple a lignments [9,23]. Several human and bovine 5¢-nucleotidases (cN-Ia, cN-Ib, cN-II, cN-III, cdN a nd m dN) were aligned by using the same approach. These proteins were also analysed for sequence motif by using the MOST program [24] with stringent cut-offs (e.g. r ¼ 0.0085). Protein secondary structure was predicted by using the PROF , SCRATCH / SSPRO and PSIPRED programs [25–27]. Identified sequence motifs were verified o n the basis of the predicted secondary structure. All sequences were further aligned by using the MACAW program [28], with minor manual adjustments. Site-directed mutagenesis Point mutants were obtained as previously described [6], with minor changes. The protocol adopted included two successive PCR reactions. In the first, a mutagenic primer was used together with a primer specific for cN-II to amplify a dsDNA fragment (megaprimer) that contained the desired mutation. Each megapr imer, purified from the agarose gel, was used in a second PCR reaction together with a second specific primer, to amplify the final nucleotide fragment, including specific sites for restriction endonucleases at the 5¢ and 3¢ terminus and, in the central part, the mutated t riplet. Once it had been cleaved, this fragment was used t o replace the corresponding one present in the expression plasmid containing bovine w ild-type cN-II. The specific for ward primers used in the PCR reactions were: NheI_F) 5¢-CCGCTAGCATGACAACC TCCTG-3¢ (from base )8 to base 14 of the pET28c-cNII construct); and AflII_F) 5¢-CAGTTGACTGGGTTCATT-3¢ (f rom base 611 to base 628). The specific reverse primers used were: KpnI_R) 5¢-AGTAGACGATGCCATGCT-3¢ (from base 982–965); and Csp45I_R) 5¢-GTTCAGCCAAGAAAATATC-3¢ (from base 1205–1186). The mutagenic primers used were as follows (the mutagenic triplette is shown in bold): M53_F) 5¢-TGGGTTTGACANHGATTATACACTTGC TGTGTA-3¢ (from base 147 to base 179) (potentially able to produce s ix different m utants: M53I, M53T, M 53N, M53K, M53S, M53R); T56_F) 5¢-TGGGTTTGACATG GATTATADNCTTGCTGTGTA-3¢ (from base 147 to base 179) (potentially able to pr oduce six different mutants: T56I, T56M, T56N, T56K, T56S, T56R); T249S_F) 4882 S. Allegrini et al. (Eur. J. Biochem. 271) Ó FEBS 2004 5¢-TTTCTTGCCTCCAACAGTGA-3¢ (from base 736 to base 755); T249V_F) 5¢-TTTCTTGCCGTCAACAG TGA-3¢ (from base 736 to base 755); S251(T/A)_F) 5¢-TGC CACCAACRCTGA CTATAA A-3¢ (from base 741 t o base 762); K292(R/M)_F) 5 ¢-GCACGGAKGC CACTGTTCT-3¢ (from base 868 to base 886); D351E_R) 5¢-C CCAAAAATGTGCTCTCCAATA-3¢ (from b ase 1065 to base 1044); D 351N_R) 5¢-CCC AAAAA TGTGCTCTCCAATA-3¢ (from base 1065 to base 1044); D356E_R) 5¢-A ATCTCCCCAAAAATGTGA TCT-3¢ (from base 1071 to base 1050); D356N_R) 5¢-AAT GTTCC CAAAAATGTGATCT-3¢ (from base 1071 to base 1050). Table 1 shows t he primer couples used to produce the mutants described in this article. The P CR mixtures and cycling c onditions were as follows. First PCR mixture: 5 0 lL containing 7.5 ng of pET28c-cNII DNA as template, 2 l M of mutagenic primer, 1 l M of specific primer, 200 l M of dNTP, 1 m M MgSO 4 and 1.25 U of Platinum Pfx DNA polymerase in P CR reaction buffer. The first PCR c ycling c onditions were: 2minat94°C; 15 s at 94 °C; 30 s a t 5 0–60 °C (dependin g on the couple of primers used); and 30 s at 68 °C. Steps 2–4 were repeated 30 ti mes. The second PCR mixture was: 25 lL containing 10 ng of pET28c-cNII DNA, all the megaprimers recovered after purification from the agarose gel (usually 0.5–0.8 l M ), 3 l M specific primer, 200 l M dNTP, 1 m M MgSO 4 and 0.7 U of Platinum Pfx DNA polymerase in PCR reaction buffer. The cycling conditions in the second PCR were the same as those used in the first PCR, but in the second PCR, the annealing temperature was always 60 °C. Expression of the recombinant proteins Bovine wild-type and recombinant cN-II mutants were prepared and purified as previously described [29]. At the N terminus all the recombinant products presented an addi- tional MGSSHHHHHHSSGLVPRGSHMAS sequence (whose amino acids were numbered with negative values) containing the histidine tag and the thrombin cleavage site. The p ro tein concentration w as determined according to Bradford [30], using BSA as a standard. The molar concentration of the enzymes was determined by using the calculated subunit molecular mass (67 300 Da). Electrophoresis and immunoblotting Electrophoresis under denaturing conditions was performed on 12% polyacrylamide gels, according to L aemmli [31]. After electrophoresis, proteins were blotted onto a PVDF membrane. Immunostaining with specific antibody was carried out as previously described [ 5]. Enzyme assays Unless stated otherwise, the nucleotidase activity of cN-II and its mutants was measured as the rate of [8- 14 C]inosine formation from 2 m M [8- 14 C]IMP in the presence of 1.4 m M inosine, 20 m M MgCl 2 ,4.5m M ATP and 5 m M dithiothre- itol, as previously described [11]. Phosphotransferase activ- ity was measured as the rate of [8- 14 C]IMP formation from 1.4 m M [8- 14 C]inosine, in the presence of 2 m M IMP, 20 m M MgCl 2 ,4.5m M ATP and 5 m M dithiothreitol, as previously described [11]. For the determination of kinetic p arameters (K m and k cat ) the concentration of t he labelled substrates ranged from 0.02 to 4 m M . A curve of dependence of the rate of phosphotransferase activity on MgCl 2 concentration wasusedtodetermineK 50 for MgCl 2 . Under these experimental conditions, the accumulation of radiolabeled inosine (nucleotidase activity) represents the sum of the phosphatase and the phosphotransferase activ- ities. It has previously been reported that, at a concentration close to the K m value (1.4 m M ), inosine reduces phosphatase activity to 50% without affecting the V max for both reactions [11]. Thus, the expected value of 2 was determined for the ratio between nucleotidase and phosphotransferase activities, under the experimental conditions used for the wild-type recombinant cN-II assay. Accordingly, an alter- ation o f t his r atio for a mutant was considered as being caused either by an alteration of the K m value for one of the two s ubstrates or by a variation of the k cat value for one of the two activities. The oxidative inhibitory effect was measured by incuba- ting the enzyme w ith CuCl 2 (final concentration 1–250 l M ) in 50 m M Tris/HCl, pH 7.4, for 10 min. Enzyme was quickly measured for nucleotidase activity, before and after the addition of 5 m M dithiothreitol to the incubation mixture. P arallel experiments were also performed by incubating cN-II with or without 20 l M 5,5¢-dithiobis- (2-nitro-benzoic acid), in 50 m M Tris/HCl, pH 7.4, at room temperature. At different time-points, samples were with- drawn and assayed for nucleotidase activity. After 80 min, Ellman’s reagen t treated-cN-II was added with 5 m M dithiothreitol and assayed f or nucleotidase activity. Structural characterization of 5¢-nucleotidase samples Purified wild-type r ecombinant 5¢-nucleotidase s amples (100 lg), obtained by treatment with or without 5 m M dithiothreitol, and with or without thrombin (1 lg), in 50 m M Tris/HCl, pH 7.4, were alkylated with 1.1 M iodo- acetamide i n 0.25 M Tris/HCl, 1.25 m M EDTA, containing 6 M guanidinium chloride, pH 7.0, at room temperature for 1 m in in the dark. Proteins were freed from s alt and excess Table 1. Primers used in PCR reactions for the production of cytosolic nucleotidase-II (cN-II) point mutants. Mut. Pr., mutant primer; Sp. Pr., specific primer; MP, megaprimer. Mutant First PCR Second PCR Mut. Pr. Sp. Pr. MP (bp) Sp. Pr. M53(I/N) M53_F + KpnI_R fi 836 + NheI_F T56R T56_F + KpnI_R fi 836 + NheI_F T249S T249S_F + Csp45I_R fi 470 + NheI_F T249V T249V_F + Csp45I_R fi 470 + NheI_F S251(T/A) S251(T/A)_F + Csp45I_R fi 465 + NheI_F K292(R/M) K292(R/M)_F + Csp45I_R fi 338 + NheI_F D351E D351E_R + AflII_F fi 455 + NheI_F D351N D351N_R + AflII_F fi 455 + NheI_F D356E D356E_R + AflII_F fi 461 + NheI_F D356N D356N_R + AflII_F fi 461 + NheI_F Ó FEBS 2004 Cytosolic 5¢-nucleotidase II mechanism (Eur. J. Biochem. 271) 4883 reagents by passing the r eaction mixtures through PD10 columns (Amersham Pharmacia Biotech), as previously reported [32]. Protein samples were manually collected, lyophilized and analysed/concentrated by SDS/PAGE under nonreducing conditions. Bands from SDS/PAGE were excised from the gel, triturated and washed with water. Proteins were in-gel digested with trypsin or 2% (v/v) formic acid, as p reviously described [33]. Digest aliquots were removed and s ubjected to a desalting/co ncentration s tep o n ZipTipC 18 devices (Milli- pore Corp., Bedford, MA, USA) before analysis by MALDI-TOF-MS. Peptide mixtures were eluted from the ZipTipC 18 in a stepwise manner, using an i ncreasing concentration of acetonitrile in the elution solution, and loaded directly on the MALDI target by using the dried droplet technique and a-cyano-4-hydroxycinnamic as mat- rix. Samples were analysed on a Voyager-DE PRO mass spectrometer (Applied Biosystems, Framingham, MA, USA). Assignments o f the reco rded mass values to individual peptides were performed on t he bas is o f t heir molecular mass and proteolytic agent specificity, as previously described [6]. Peptide mixtures were also fractionated by RP-HPLC on a Vydac 218TP52 column (250 · 2.1 mm), 5 lm, 300 A ˚ pore size ( The Separation Group, Hesperia, CA, USA) by using a linear, 5–60% gradient of acetonitrile in 0.1% (v/v) trifluoroacetic acid over 60 min, at a flow rate of 0.2 m LÆmin )1 . Individual components were collected manu- ally. Disulfide-containing peptides were identified on the basis of their mass value. Sequence analysis was performed by using a Procise 491 protein sequencer (Applied Biosys- tems) eq uipped with a 140C microgradient HPLC and a 785 A UV detector (Applied Biosystems) for the identifica- tion of PTH a mino acids. Results cN-II and HAD superfamily CN-II chemical labelling a nd site-directed mutagenesis experiments identified D52 as a residue that is essential for enzyme activity and involved in phosphate-adduct forma- tion [6]. This amino acid occurs in a sequence region similar to motif I , which is common to all members of the HAD superfamily [8]. Iterated sequence comparisons and posi- tion-specific iterated searches starting from bovine cN-II or different phosphomonoesterases, phosphotransferase s, phosphomutases and dehalogenases were used to identify, in the cN-II primary structure, the remaining two motifs already reported for these proteins. Similarly to HAD superfamily members, these motifs should contain amino acids h ypothetically present in the enzyme active site which are essential f or metal ion coordination, nucleophilic attack to substrate and stabilization of an excess of negative charge in the reaction intermediate. Furthermore, analysis of HAD superfamily members demonstrated that conserved residues from each of the motifs appear to occur at specific positions in the succession of secondary structure elements [9]. For this reason, the sequence of cN-II was also analysed in order to predict the secondary structure of the protein. In addition to the already identifie d motif I, this investigation highligh- ted two separate regions in the cN-II primary structure as being a ssociated with motif II and motif III (Fig. 1). As Fig. 1. Multiple alignment of mammalian 5¢-nucleotidases and members of the P-type ATPase-L-2-haloacid dehalogenase (ATPase-HAD) super- family. Proteins are listed under their SWISS-PROT codes. Bb, Bos bovis;Ec,Escherichia coli;Eh,Enterococcus hirae;Hs,Homo sapiens;Mg, Mycoplasma genitalium;Psp,Pseudomonas sp.; Sa, Staphylococcus aureus;Sc,Saccharomyces cerevisiae;andSp,Schizosaccharomyces pombe. Only the t hree common sequence motifs are reported. The numbers in dicate the distances to the N term inus of each protein and the sizes of the gaps between aligned s egment s. Th e upper, mid dle and lower block of sequences i nclude mammalian 5¢-nucleotidases, members of the HAD superfamily and P-type ATPases, respectively. Blue shading indicates conserved amino acid residues required for catalytic activity. Red shading indicates conserved a mino acids alternatively present in motif I. Yellow shading indicates uncharged amino acid r esidues. Common secondary s tructure elements are indicated as a-helices, b-strand s and l (loop) regions. 4884 S. Allegrini et al. (Eur. J. Biochem. 271) Ó FEBS 2004 clearly illustrated in the reported multiple alignment, conserved amino acids in each o f the motifs were inserted in regions that always presented uncharged residues at specific positions and w ith a well-defined secondary struc- ture content. Moreover, on the basis of the recent obser- vation that md N also belongs to the HAD s uperfamily, the above reported sequence analysis was extended to all mammalian 5¢-nucleotidases. I terated s equence c ompari- sons and position-specific iterated searches identified the three motifs in all of these proteins, except for eN, (Fig. 1) suggesting that cytosolic and mitochondrial 5¢-nucleotidases and deoxynucleotidases present a structural arrangement related to the HAD sup erfamily fold. Site-directed mutagenesis To verify the p redicted role for the conserved residues reported in Fig. 1, 13 mutated cN-II products were constructed and expressed. Extracts were prepared 16 h after the addition of isopropyl thio-b- D -galactoside (IPTG) and recombinant wild-type and cN-II mutants were purified as previously reported [6]. In all cases, SDS/PAGE analysis showed a single component migrating with an apparent molecular mass of 60 kDa (data not shown). Purified proteins were used in kinetic measurements, and their parameters are reported in Table 2. The fact that all the expressed p roteins conserved the same chromatographic behaviour (data not shown) indicated that observed changes of activity were not cau sed by gross folding problems [34]. In a previous work [6], we demonstrated that both conservative and nonconservative substitution of D52 and D54 (motif I) completely a bolished b oth e nzyme a ctivity and formation of the cN-II–phosphate intermediate. Replacement at other positions of this motif strongly affected how cN-II functioned. In fact, substitution at position 56 (mutant T56R) resulted in a protein devoid of nucleotidase and phosphotransferase activities. On the other hand, the mutagenesis of M53 h ad a l ess severe effect. However, while nonconservative substitutions (M53N) resulted in a strong decrease in catalytic efficiency, conser- vative substitution (M53I) led t o e ffects o n b oth enzyme activity and affinity towards IMP. This latter mutant exhibited the lowest n ucleotidase vs. phosphotransferase activity ratio ; it also showed a sigmoid dependence on Mg 2+ concentration. These results indicate the essential role of these residues for proper D52 and D54 orientation and effective c N-II catalysis, confirming the function of motif I, as deduced b y previous c hemical labelling and site-directed mutagenesis experiments. The conserved amino acid present in motif II, which is common to all HAD superfamily members, is always a serine or a t hreonine residue. T his amino acid is important for a correct orientation of the substrate within the active site through specific hydrogen bonding. The alignment reported in Fig. 1 shows that T249 is an essential residue for cN-II activity. However, another amino acid (S251), with similar properties, occurs closely i n m otif II. In order to unambiguously identify the residue present in motif II , conservative and nonconservative mutants of both amino acids were p repared. Mutant T249V showed a strongly reduced enzyme activity and an alteration of K m values for both substrates. On the other hand, a conservative substi- tution (mutant T249S) yielded a kinetic behaviour more similar to that of wild-type e nzyme. In addition, the effect on enzyme ac tivity exerted by a nonconservative mutation of residue 251 (mutant S251A) was far less pronounced than that produced by the conservative S251T mutation. These results demonstrated the essential role of the T249 hydroxyl group for cN-II catalysis, thus confirming the nature of motif II deduced by multiple sequence alignments. Motif III in the HAD superfamily is characterized by the presence of a conserved lysine residue and two negatively charged residues which are involved in stabilizing the negatively charged reaction intermediate and metal ion Table 2. Effect of point mutations on various kinetic parameters of bovine recombinant cytosolic nucleotidase-II (cN-II). Nucleotidase and phos- photransferase activities were measured as described in the Experimental procedures. The results reported are the average of at least three independent assays. IMP, inosine monophosphate; nd, not detectable; ND, not determined; WT, wild type. Mutant k cat Phosphotransferase (s )1 ) Nucleotidase/ phosphotransferase K m IMP (m M ) K m inosine (m M ) k cat /K m inosine K 50 MgCl 2 (m M ) WT 40.0 ± 13 1.8 ± 0.1 0.1 ± 0.04 1.1 ± 0.3 36.0 ± 2.4 1.8 ± 0.6 Motif I M53I 3.7 ± 2.1 0.9 ± 0.05 1.0 ± 0.4 0.8 ± 0.05 4.5 ± 2.3 3.0 ± 1.0 sigmoid M53N 0.6 ± 0.1 2.4 ± 0.1 0.1 ± 0.01 1.1 ± 0.4 0.6 ± 0.1 ND T56R nd nd nd nd nd nd Motif II T249S 19.8 ± 1.9 3.4 ± 0.2 0.2 ± 0.06 0.9 ± 0.1 22.7 ± 1.2 4.1 ± 1.1 T249V 0.3 ± 0.15 1.5 ± 0.1 0.4 ± 0.25 0.25 ± 0.07 1.1 ± 0.3 4.4 ± 1.2 S251T 3.4 ± 0.6 6.7 ± 0.3 0.3 ± 0.15 1.5 ± 0.5 2.3 ± 0.4 3.5 ± 1.8 S251A 12.3 ± 1.3 3.7 ± 0.2 0.1 ± 0.01 1.0 ± 0.15 11.8 ± 0.5 3.5 ± 1.3 Motif III K292R nd nd nd nd nd nd K292M < 0.1 1.8 ± 0.1 ND 0.5 ± 0.1 < 0.2 ND D351E < 0.1 1.5 ± 0.1 ND 0.9 ± 0.2 < 0.1 > 30 D351N nd nd nd nd nd nd D356E 2.2 ± 1.0 2.5 ± 0.1 0.2 ± 0.1 1.0 ± 0.25 2.2 ± 0.8 15.0 ± 4 D356N 1.4 ± 0.1 3.2 ± 0.2 0.2 ± 0.06 10.2 ± 0.8 0.1 ± 0.02 > 30 Ó FEBS 2004 Cytosolic 5¢-nucleotidase II mechanism (Eur. J. Biochem. 271) 4885 coordination, respectively. As expected, on the basis of the proposed alignment, the mutation of K292 (mutant K292R and K292M) strongly affected cN-II activity. Similarly, t he mutation of two aspartate residues (D351 and D356) resulted in very poor nucleotidase and phosphotransferase activities and a significantly reduced affinity towards Mg 2+ . Conservative mutations (mutant D351E and D3 56E) had a less pronounced effect than nonconservative mutations. Furthermore, D356N show ed a 10-fold increase in the K m value for inosine, suggesting a possible role for this amino acid in the interaction with the second substrate. All these data confirmed the nature of the residues p resent in motif III, as deduced by sequen ce alignment. A general comparison of all the kinetically characterized mutants showed that the nucleotidase vs. phosphotrans- ferase activity ratio was significantly altered in two cases: mutant S251T and mutant M53I . In the first case, this phenomenon might be caused by a decrease of phospho- transfer efficiency, as alterations of the K m value measured for both s ubstrates were very slight. In the latter case, the value observed for this parameter was in line with a 10-fold increase of the K m for IMP. Sensitivity to oxidizing conditions It has been reported that freshly purified calf thymus cN-II displays full activity only i n the presence of dithiothreitol [5,11,12], suggesting that ox idation may modulate enzyme properties. To confirm this observation, recombinant wild- type cN-II was incubated with different concentrations of CuCl 2 , and its remaining activity was measured before a nd after the addition of dithiothreitol to the reaction mixture. The results reported in Fig. 2 A demonstrate that CuCl 2 treatment inhibited the enzyme in a concentration-depend- ent manner. The loss of activity was reverted by the presence of the reducing agent. Recombinant wild-type cN-II was also treated with a different oxidizing agent, 5,5¢-dithiobis- (2-nitro-benzoic acid), and its remaining activity was measured. The results shown in Fig. 2B clearly demonstrate that cN-II was str ongly sensitive t o this r eagent. A fter 80 min of incubation, dithiothreitol was added to the reaction mixture, resulting i n a complete recovery of activity, t hus demonstrating that 5,5¢-dithiobis-(2-nitro- benzoic acid) oxidation can be reverted by reducing agents. Proteolytic generation of two cN-II forms In a previous work, w e observed t he simultaneous presence of two cN-II forms in preparations from calf thymus, distinguishable f or electrophoretic, c hromatographic and regulatory properties [35]. In fact, the cN-II species (form B) with faster electrophoretic mobility (54 kDa) was activated by ADP and BPG, and a synergistic stimulatory effect of these compounds was also observed. On the other hand, the slower migrating species (form A) (59 kDa), was activated to a greater extent by ADP and BPG, and the synergistic effect was absent [35]. T o a scertain whether form B was arising from an intracellular proteolytic event or from a degradative process during preparation, freshly isolated tissues were solubilized directly in hot sample buffer for SDS/PAGE, and the extracted proteins were analysed by Western blotting following SDS/PAGE (Fig. 3A). Two major immunoreactive polypeptides migrating at 54 and 59 kDa were d entified, thus demonstrating that the 54 kDa species is not generated b y a preparation artefact. We also noted that highly purified recomb inant cN-II preparations, although stable for activity, degraded very slowly to enzyme forms with a lower apparent molecular mass (results not shown). Furthermore, w hen a freshly prepared recombinant product (60 kDa) was incubated with thrombin to remove the His-tag-containing sequence, different polypeptide species were obtained, depending on the experimental conditions. In addition to the expected 59 kDa polypeptide, overnight incubation with thrombin at 25 °C induced production of a cN-II form with an apparent molecular mass o f 54 kDa (Fig. 3B), while overnight incubation with thrombin at 4 °C only gave rise to the expected 59 kDa protein. Parallel experiments with native cN-II from calf thymus, containing both form A and form B, demonstrated that thrombin treatment induced increased Fig. 2. Effect of CuCl 2 and 5,5¢-dithiobis-(2-nitro-benzoic acid) treat- ment on the activity of cytosolic nuc leotidase-II (cN-II). Purified wild-type recombinant cN-II (1 l M ) was incubated with different concentration s of CuCl 2 , for 10 min, at room temperature (A). The rate of ino sine mon ophosphate (IMP) hydrolysis was measured, as described in the Experimental procedures, b efore (square) and after (circle) the addition of 5 m M dithiothreitol to the incubation mixture. Similarly, wild-type recombinant c ytoso lic nucleotidase-II (cN-II) (1 l M ) was incubated with (square) or without (circle) 20 l M 5,5¢-dithiobis-(2-nitro-benzoic acid ), at room temperature, and cN-II activity was measured as described in the Exp erimental p rocedures (B). After 80 min, the Ellman’s reagen t treated-en zyme was added with 5m M dithiothreitol and IMP hydrolysis was measured. 4886 S. Allegrini et al. (Eur. J. Biochem. 271) Ó FEBS 2004 amounts of form B, thus yielding a polypeptide with an apparent molecular m ass of 54 kDa co-migrating with that obtained from the recombinant enzyme (Fig. 3B). These results suggest that an unpredicted site of protease cleavage is present in the primary structure of cN-II, in addition to the predicted site present at t he N terminus of the recombinant product. To demonstrate definitively that the 59 kDa and 54 kDa species obtained from the recombinant enzyme were similar to those occurring in calf thymus cN-II, their sensitivity towards different regulatory ligands was investigated. A s a lread y reported for form B from calf thymus, the 54 kDa form purified from thrombin-treated recombinant cN-II w as a ctivated by ADP a nd BPG (Fig. 4A). Also in this case, the reported synergistic stimulatory effect of thes e compounds was observed; in fact, a K 50 value for ADP of 4 m M and 1 m M was measured in the absence and in the presence o f 200 l M BPG, respectively. Similarly t o form A from calf thymus, t he slower migrating recombinant species (59 kDa) was activa- ted to a greater extent by A DP and BPG, and the reported synergistic effect was absent (Fig. 4B). In fact, a K 50 value for A DP of 2.5 m M was measured e ither in the presence or absence of 200 l M BPG. Sequencing analysis of all species reported in Fig. 3B yielded the same N-terminal sequence, thus indicating that this proteolytic event occurred at the protein C terminus. Hence, a still-unknown intracellular proteolytic process, which removed a C-terminal polyp ep- tide, wo uld seem to control the relative abundance of these two cN-II forms, thus modulating the different response to enzyme ligands. Interestingly, in addition to the above-mentioned differ- ences in regulatory properties, the two cN-II forms gener- ated by thrombin treatment presented significant differences also in their activity a s a function of dithiothreitol concen- trations (results not shown). In fact, the 59 kDa species obtained from recombinant wild-type cN-II, similarly to the unprocessed product, was highly sensitive to dithiothreitol. On the contrary, the 54 kDa species was fully active, even in the absence of reducing agents. Structural characterization of 5¢-nucleotidase forms In order t o determine the unexpected site for t hrombin hydrolysis and to identify possible r esidues involved in cN-II redox regulation, various protein samples were subjected or not subjected to the reductive conditions used in the enzymatic assay, and digested or not digested with thrombin, and then alkylated with iodoacetamide under denaturing nonreducing c onditions. Following the r eac- tion, the samples were desalted and analysed by SDS/ PAGE, and in each case yielded a single component. The excised bands were digested either w ith trypsin o r 2% (v/v) formic acid a nd the c orresponding peptide m ixtures were analysed by MALDI-TOF-MS or resolved by RP-HPLC and further characterized. Table 3 summarizes the results obtained; in all cases, mass spectrometry experiments allowed a complete structural characterization of the analysed species. As expected, in th e sample prepared under reductive conditions and in the absence of thrombin, signals at m/z AB Fig. 3. Immunob lotting characterization o f different cyto solic nucleotidase-II (cN-II) forms. (A) I mmunoblotting analysis of fresh bovine calf thymus tissues d irectly homogenized and extract ed in a hot sample buffe r for SDS/PAGE. The analysis was performed on 10 lg of total pro teins. (B) Immunoblotting analysis of partially purified calf thymus and purified wild-type recombinant cN-II samples following incubation with thrombin. Lanes 1–3: 3 lgofcalfthymuscN-IItreatedasfollows:lane1,keptat4°C; lane 2, incubated overnight at 25 °C; lane 3, incubated overnight at 25 °C in the presence o f thrombin. Lanes 4–6: 3 lg of wild-type recombinant e nzyme treated as follows: lane 4, k ept at 4 °C; lane 5, incubated overnight at 25 °C; lane 6, incubated overnight at 25 °C in the presence of thrombin. AB Fig. 4. Regulatory effect of ADP and 2,3-biphosphoglycerate (BPG) on the different cytosolic nucleotidase-II (cN-II) forms g enerated following thrombin treatment of the wild-type recombinant enzyme. Enzyme assays were performed in the absence of ATP, as described in the Experimental procedures. (A) Twenty-five nanograms of the 54 kDa form were assayed with a variable amount of ADP, in the presence (open symbols) or absence (closed symbols) of 200 l M BPG. (B) Twenty-five nanograms of the 59 kDa form were assayed with variable amounts of ADP, in the presence (open symbols) or absence (closed s ymbols) of 200 l M BPG. Inserts show the SDS/PAGE ana- lysis of the respective enzyme species. Ó FEBS 2004 Cytosolic 5¢-nucleotidase II mechanism (Eur. J. Biochem. 271) 4887 1093.2, 1731 .2, 2052.1, 20 66.7, 2178.4, 2180.3, 2968.5, 3457.2, 3502.4, 4023.6 (trypsin) and 2131.4, 2920.2, 3035.4, 3242 .8, 3252.6, 37 28.5, 3813.3, 3928.7, 3944.8, 4001.1, 4334.9 (2% formic acid), corresponding to the carboxyamidomethylated peptide species, clearly demon- strated that all eight cysteine residues occurring in the polypeptide chain were present in a reduced state. On the other hand, the cN-II sample prepared in the presence of reducing agents and thrombin treatment, in addition to the mass signals reported above, showed the occurrence of clear MH + signals at m/z 7363.4 (trypsin) and 5742.8, 5959.1, 6349.4 (2% formic acid) that were tentatively assigned to S-S-containing peptides (Table 3). T hese peaks suggested the occurrence in this sample of an oxidized cN-II f orm containing the d isulfide bridge C175-C547, in addition to a fully reduced enzyme species. This hypothes is w as con- firmed by Edman degradation analysis of the purified disulfide-containing peptides. I n fact, the t ryptic peptide (MH + at m/z 7363.4) revealed the occurrence of PTH-Cys- carboxyamidomethylated (PTH-Cys-CAM) at position 167 and P TH-cystine at pos ition 175, together with the absence of any PTH amino acids at position 547 [36]. Similarly, sequencing analysis of the acid-generated peptides (MH + at m/z 5742.8, 5959.1, 6349.4) demonstrated the presence of PTH-Cys-CAM at position 181 and PTH-cystine at posi- tion 547, together with the absence of any PTH amino acids at position 175 [36]. These results definitively d emonstrate the nature of the oxidized enzyme and the extreme sensitivity of C175 and C547 to changes in redox conditions. Different data were generated from the MALDI-TOF- MS analysis of a cN-II sample obtained f ollowing pro- longed thrombin treatment. This species migrated with an apparent mass of 54 kDa. The occurrence in t he spectr a of new signals at m/z 1101.8, 1318.5, 1398.7, 1709.2, 2167.8, 3623.7 (2% formic acid), as well as the disappearance of the signals at m/z 1901.3, 4023.6 (trypsin) and 3280.6, 3728.5, 3944.8, 4334.9 (2% formic acid), corresponding to the N- and C -terminal r egion o f t he intact protein, clearly demonstrated that, in addition to the expected site (R-6) present at t he N t erminus, cN-II was hydro lysed by thrombin also at R526 (Table 3). C onsequently, in t he spectrum there were no mass signals corresponding to peptides containing the d isulfide bridge C175–C547. These data were in perfect agreement with the above-mentioned insensitivity o f the thromb in-generated 54 kDa species to the o xidative conditions. In f act, as thrombin-treated cN-II is devoid of the C-terminal p eptide containing C547, involved in the S -S bridge , i t i s no l onger sensitive t o changes in redox conditions. Discussion The HAD fold defines a versatile hydrolase/mutase/trans- ferase superfamily which a ppears to f unction on the Table 3. MALDI-TOF-MS analysis of air-exposed, reduced and thrombin-treated cytosolic nucleotidase-II (cN-II) samples. Protein s ample s su b- jected or not subjected to the reductive conditions used in enzymatic assay, and digested or not digested with thrombin, w ere alkylated with iodoacetamide in denaturing, nonreducing conditio ns and separated by S DS/PAGE under no nreduc ing conditions. B ands were digeste d in situ with trypsin or 2% (v/v) formic acid and peptide extracts were analysed by MALDI-TOF-MS. For simplicity, the table only reports the carboxy- amidomethylated (CAM), disulfide-linked (S-S) and N-/C-terminal peptides. The mass is reported as average values. Trypsin Peptide 2% (v/v) Formic acid Peptide Native cN-II MH + (m/z) Reduced cN-II MH + (m/z) Thrombin- treated cN-II MH + (m/z) Native cN-II MH + (m/z) Reduced cN-II MH + (m/z) Thrombin- treated cN-II MH + (m/z) 634.6 634.7 634.2 (35–39) 1101.8 (519–526) 654.8 654.9 654.7 (522–526) 1318.5 (517–526) 675.7 675.4 675.9 (30–34) 1398.7 ()5–7) 1093.5 1093.2 1093.0 (178–186)CAM 1709.2 (514–526) 1433.3 1433.6 1433.5 (516–526) 1934.3 1934.1 1934.6 (497–513) 1475.4 1475.4 1475.8 (510–521) 2131.2 2131.4 2131.7 (171–187)CAM 2 1554.6 1554.7 1554.1 ()5–8) 2324.9 2324.2 2324.5 (497–516) 1730.9 1731.2 1730.6 (48–61)CAM 2920.7 2920.2 2920.7 (147–170)CAM 1901.0 1901.3 ()23–6) 3035.3 3035.4 3035.7 (146–170)CAM 2052.2 2052.1 2052.6 (426–442)CAM 3242.5 3242.8 3242.9 (307–337)CAM 2067.0 2066.7 2066.9 (112–129)CAM 3252.9 3252.6 3252.4 (432–459)CAM 2178.2 2178.4 2178.2 (178–195)CAM 3280.7 3280.6 ()23–7) 2180.8 2180.3 2180.9 (425–442)CAM 3623.7 (497–526) 2968.3 2968.5 2968.1 (314–342)CAM 3728.3 3728.5 (519–549)CAM 3253.5 3253.6 3253.9 (479–507) 3813.1 3813.3 3813.6 (114–145)CAM 3456.7 3457.2 3457.0 (150–177)CAM 2 3928.5 3928.7 3928.3 (114–146)CAM 3502.1 3502.4 3501.9 (99–129)CAM 3944.5 3944.8 (517–549)CAM 4023.4 4023.6 (527–560)CAM 4000.8 4001.1 4000.5 (21–52)CAM 7363.4 (150–177)CAM + (527–560)S-S 4335.2 4334.9 (514–549)CAM 5742.8 (171–187)CAM + (519–549)S-S 5959.1 (171–187)CAM + (517–549)S-S 6349.4 (171–187)CAM + (514–549)S-S 4888 S. Allegrini et al. (Eur. J. Biochem. 271) Ó FEBS 2004 common scheme of an e nzyme-aspartate ester formation followed by the transfer of the ester group to a nucleophile molecule, i.e. water for hydrolases and dehalogenases, a different chemical m oiety o n the initial substrate for mutases, or a second substrate for transferases. With the sole exception of dehalogenases, all family members require Mg 2+ ion in the active site to promote covalent intermediate formation. Enzyme functionalities involved in catalysis are conserved within members. They appear to be juxtaposed in space, as determined on the basis of the crystallographic structures solved to date [7,9,37,38]. This determines the occurrence of three specific sequence motifs conserved among all superfamily members [9,23]. On the other hand, the utilization of the HAD fold for diverse functions is demonstrated by its rearrangement in a variety of topolo- gical variants [38]. In all cases, catalysis always involves residues present in an a/b Rossmann-fold domain c onsisting of a centrally located p arallel b-sheet surrounded by a-helices. Depending on the nature of t he enzyme, t his core presents large helical insertions which lead to additional noncatalytic domain(s ). E xcluding the three conserved motifs, the level of sequence divergence a mong proteins belonging t o the HAD superfamily is so elevated (sequence identity < 10%) that the relationship between the family members would go unrecognized. This means that some members of t his family may not yet have been identified, as a result of the limitation in the approaches used for sequence database search and/or various molecular dimensions of the analysed polypeptides. In our opinion, this is true for the six mammalian 5¢-nucleotidases reported in F ig. 1. This hypo- thesis, originally proposed by our laboratory [6], has been recently c onfirmed by the resolution of the crystallographic structure of mdN [7]. The characterization of the reaction mechanism [5], the absolute requirement of a Mg 2+ ion [11,12], the detection of a phosphorylated intermediate involving the first aspartate of its DMDYT motif [6] and the identification of the three conserved motifs reported in this study, all strongly support the idea t hat cN-II also belongs to the HAD superfamily. Mutagenesis studies at specific amino acid positions predicted b y the reported alignment allowed us to identify a series of residues essential for cN-II c atalysis. In fact, the modification of m easured kinetic parameters caused by conservative and nonconservative substitutions suggested a specific role of these amino acids in the cN-II active site. Our results are perfectly in line with t hose already reported for phosphoserine phosphatase [8,39] and Ca 2+ -ATPase [40– 42], thus demonstrating that cN-II presents a catalytic machinery which very much resembles those of the other members of the HAD superfamily. Table 4 summarizes the results obtained for these three enzymes with mutants at equivalent positions. Similarly to phosphoserine phosphatase (PSP) and Ca 2+ - ATPase, mutations of the two aspartates in motif I (D52 and D54) totally abolished cN-II activity. The first residue is directly responsible for the formation of the enzyme– phosphate intermediate [6], and the second would seem to be involved in adduct stabilization and Mg 2+ ion coordi- nation, as already observed for the other two enzymes [9,38]. Conservative mutations at cN-I I D54 had a more pro- nounced effect than those observed a t t he cor responding residues in phosphoserine phosphatase and Ca 2+ -ATPase, thus emphasizing the important role of this amino acid for effective c N-II c atalysis. In addition, our e xperiments demonstrated, for the first time, that mutations at other conserved positions of motif I also affect enzyme function- ing ( Fig. 1) (Table 2). In fact, substitution at position 56 (mutant T56R) resulted in a protein totally devoid of nucleotidase and ph osphotransferase activity. Even though this effect might be caused by differences in relative steric hindrance, this conserved threonine residue has been reported t o establish a specific h ydrogen bond e ssential for a correct positioning of the active site nucleophile in the structure of 2 -haloacid dehalogenases and phosphonoacet- aldehyde hydrolases [43]. S imilarly, kinetic analyses o f M53 mutants d emonstrate that replacements in this position can also affect cN-II catalysis, probably by influencing the correct orientation of the two aspartate residues. On the other hand, the clear similarity observed in the effect of conservative and nonconservative m utations at T249, with respect to S109 in phosphoserine phosphatase and T625 in Ca 2+ -ATPase, identified this amino acid as the cN-II r esidue of motif I I e ssential f or enzyme catalysis (Table 4). These results are in line with t he hypothesis that the h ydroxyl group of T249 is implicated in stabilization of the covalent intermediate, as already demonstrated for the corresponding residues of motif II in mdN, HAD and PSP [7,37,38]. Finally, the effect of mutations at the conserved lysine and the two negatively c harged residues putatively present i n cN-II motif III paralleled well with those observed for phosphoserine phosphatase and Ca 2+ -ATPase (Table 4). A comparison of the kinetic parameters observed for these proteins suggests t hat, in cN-II, K292 is the basic amino acid essential for the stabilization of the negatively Table 4. Comparison of the effect of mutations on the activity o f three different enzymes of the L-2-haloacid dehalogenase (HAD) superfamily. The values reported indicate the relative activity with respect to the wild-type enzyme. Residues in equ iva len t positio ns in Fig. 1 are in the same row. Results a re taken from the following references: cytosolic nucleotidase-II (cN-II), this article and [6] phosphoserine phosphatase [8], and [29] Ca 2+ ATPase [30–32]. PSP, phosphoserine phosphatase; WT, wild type; low exp, low e xpression. cN-II PSP Ca 2+ -ATPase Mutation Activity Mutation Activity Mutation Ca 2+ transport WT 100 WT 100 WT 100 Motif I D52E 0 D20E 0 D351E 0 D52A 0 D20N 0 D351N 0 D54E 0 D22E 50 T353S 20 D54A 0 D22N 0 T353A 0 Motif II T249S 20 S109T 115 T625S 79 T249V 1.6 S109A 6 T625A low exp Motif III K292R < 0.1 K158R 1 K292M 0.1 K158A < 0.4 D351E 0.7 D179E 78 D703E 31 D351N < 0.1 D179N 0.6 D703N < 5 D356E 2.3 D183E 63 D707E < 5 D356N 0.6 D183N < 0.4 D707N < 5 Ó FEBS 2004 Cytosolic 5¢-nucleotidase II mechanism (Eur. J. Biochem. 271) 4889 charged reaction i ntermediate. On the other hand, D351 and D356 seem to be involved in metal ion coordination, as substitutions at these positions completely abolished enzyme activity and caused a n increase i n the Mg 2+ K 50 values. However, i f conservative mutations at the corres- ponding residues in PSP and Ca 2+ -ATPase generated enzyme species still presented a certain residual activity, these mutations in cN-II strongly affecte d enzyme catalysis suggesting that, a s already observed for motif I, Mg 2+ coordination in cN-II strictly requires aspartate residues. Therefore, according to the mutagenesis and chemical labelling experiments reported above, the active site of cN-II should be similar to that schematically represented in Fig. 5. A careful comparison of the sequence a nd secondary structure elements with other 5¢nucleotidases and members of the HAD superfamily revealed that cN-II, in addition to the canonical a/b core domain responsible for enzyme catalytic activity, presents a large noncatalytic extension at its C terminus. A differential processing of this domain identifies the two naturally occurring cN-II forms that present a distinct response to the regulatory properties exerted b y v arious ligands and o xidizing reagents . T his phenomenon was originally hypothesized by us [35] and is now demonstrated in the work presented above by a biochemical and structural characterization of the 54 and 59 kDa species observed in calf thymus or generated by thrombin treatment of the wild-type recombinant product. These results would seem to show that the cN-II C-terminal domain is probably involved in the modulation of enzyme activity, although t he fine structural details associated with this regulation have not yet been elucidated. A s till- unknown protease present in calf thymus and other cells should cleave intact cN-II closely to R526, thus generating the two differently regulated enzyme forms. Their s imulta- neous presence should make the enzyme less susceptible not only to physiological variations in cell energy charge, but also to fluctuations of cellular redox status, as demonstrated by the results reported in this work. In conclusion, the essential residues involved in the catalysis and regulation of the cN-II-assisted hydrolysis/ phosphate transfer of purine monophosphates were ascer- tained by a combined mutagenesis/proteomic i nvestigation. An extended structure-based sequence alignment of 5¢-nucleotidases provided support for a common structural and m echanistic origin of these enzymes, revealing a strong relationship to the HAD superfamily. W e are currently studying the crystallographic structure of the two naturally occurring forms of cN-II, in the presence or absence of various nucleotide analogs. These studies should be able to ascertain the 3D details responsible for the enzyme prop- erties reported in this work. 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Mechanistic studies on bovine cytosolic 5¢-nucleotidase II, an enzyme belonging to the HAD superfamily Simone Allegrini 1, *, Andrea Scaloni 2, *,. conserved among all superfamily members [9,23]. On the other hand, the utilization of the HAD fold for diverse functions is demonstrated by its rearrangement