Mechanistic studies on bovine cytosolic 5'-nucleotidase II, an enzyme belonging to the HAD superfamily
| Autor: | Maria Giovanna Careddu, Rossana Pesi, Simone Allegrini, Lino Ferrara, Marcella Camici, Maria Grazia Tozzi, Andrea Scaloni, Chiara D'Ambrosio, Giovanna Cuccu |
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| Rok vydání: | 2004 |
| Předmět: |
Protein Conformation
Molecular Sequence Data Sequence alignment Biology Biochemistry Phosphotransferase Cytosol Nucleotidase Animals Amino Acid Sequence Site-directed mutagenesis 5'-Nucleotidase Magnesium ion DNA Primers Dehalogenase chemistry.chemical_classification Base Sequence Sequence Homology Amino Acid Hydrolysis Amino acid chemistry Mutagenesis Site-Directed Cattle Electrophoresis Polyacrylamide Gel Cysteine |
| Zdroj: | European Journal of Biochemistry. 271:4881-4891 |
| ISSN: | 1432-1033 0014-2956 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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