Autophosphorylation of Solanum chacoense cytosolic nucleoside diphosphate kinase on Ser117.
NDPK catalyses the interconversion of NTPs and NDPs using a phosphohistidine intermediate as part of its catalytic site. Recombinant Solanum chacoense cytosolic NDPK incubated with [gamma-(32)P]ATP was allowed to autophosphorylate and (32)P-labelled P-Ser was identified in an acid hydrolysate of the protein by two-dimensional TLC. Further analysis of (32)P-labelled recombinant NDPK by tryptic digestion followed by automated Edman sequencing of the radioactive peptide allowed the identification of a single and conserved P-Ser residue at position 117. Analysis of site-directed mutants where Ser117 was substituted to Asp indicated that the presence of a negative charge at position 117 dramatically lowered the enzyme's catalytic efficiency. Ser autophosphorylation was markedly reduced with increasing ADP concentrations in the autophosphorylation assay. These findings provide evidence that autophosphorylation of cytosolic NDPK on Ser117 could constitute a regulatory mechanism for this important enzyme and that autophosphorylation of Ser117 is modulated by NDP availability.
Dorion
Sonia
S
IRBV, Université de Montréal, 4101 Rue Sherbrooke est, Montréal, Quebec, H1X 2B2 Canada.
Dumas
France
F
Rivoal
Jean
J
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D012694:Serine / Q000737:chemistry*; D032322:Solanum / Q000201:enzymology*
D010940:Plant Proteins; D020033:Protein Isoforms; D012694:Serine; D006639:Histidine; D009701:Nucleoside-Diphosphate Kinase