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Cytosolic 5¢-nucleotidase/phosphotransferase specific for 6-hydroxypurine monophosphate derivatives (cN-II), belongs to a class of phosphohydrolases that act through the formation of 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 conservedmotifswereperformed. | Eur. J. Biochem. 271 4881-4891 2004 FEBS 2004 doi 10.1111 j.1432-1033.2004.04457.x Mechanistic studies on bovine cytosolic 5 -nucleotidase II an enzyme belonging to the HAD superfamily Simone Allegrim1 Andrea Scaloni2 Maria Giovanna Careddu1 Giovanna Cuccu3 Chiara D Ambrosio2 Rossana Pesi3 I Marcella Camici3 Lino Ferrara2 and Maria Grazia Tozzi3 1 Dipartimento di Scienze del Farmaco Universita di Sassari Italy 2Proteomics and Mass Spectrometry Laboratory ISPAAM National Research Council Naples Italy 3Dipartimento 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 the formation of an enzyme-phosphate intermediate. Sequence alignment with members of the P-type ATPases L-2-halo-acid dehalogenase superfamily identified three highly conserved 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 measured kinetic parameters caused by conservative and nonconservative substitutions suggested that motif I is involved in the formation and stabilization of the 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 catalysis. 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 superfold. Structural determinants involved in differential regulation by nonprotein ligands and redox reagents of the two naturally occurring cN-II forms generated by proteolysis were ascertained by combined biochemical and mass .