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 cyto-solic NDPK incubated with [g-32P]ATP was allowed to autophosphorylate and 32P-labelled P-Ser was identi-fied in an acid hydrolysate of the protein by two-dimensional TLC. Further analysis of 32P-labelled recombinant NDPK by tryptic digestion followed by automated Edman sequencing of the radioactive pep-tide 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 autophosphory-lation 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 mod-ulated by NDP availability. Key words: Enzymology, nucleoside diphosphate kinase, plant, phosphoamino acid, phosphorylation, protein sequencing, site-directed mutagenesis