Mechanistic studies on bovine cytosolic 5′‐nucleotidase II, an enzyme belonging to the HAD superfamily

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‐haloacid 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 spectrometric investigations. These experiments indicated that the C‐terminal region 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 as regulatory phenomena.