PHOSPHORYLATION MECHANISM OF NUCLEOSIDE DIPHOSPHATE KINASE - P-31-NUCLEAR MAGNETIC-RESONANCE STUDIES

The phosphorylation mechanism of Dictyostelium discoideum nucleoside diphosphate (NDP) kinase was investigated by NMR. P-31 chemical shifts were measured on both native and denatured enzyme. In the enzymatically phosphorylated enzyme denatured by 9 M urea or 7 M guanidine hydrochloride, the NDP kinase phosphohistidine signal appeared between the signals of N delta and N epsilon free monophosphohistidines used as reference compounds and added to the sample. A signal with the same intermediate position was also observed in the pronase digest of the alkaline-denatured phosphorylated enzyme. However, when phosphohistidines of the phosphorylated synthetic peptide pGlu-His-Gly were taken as references, the NDP kinase and the N delta peptide phosphohistidine signals were shown to be identical, providing evidence that phosphorylation occurs on the N delta of the active site histidine residue. Moreover, the rate of hydrolysis of the histidine-bound phosphate is in agreement with a modification at the N delta position. Phosphorylation of the NDP kinase by phosphoramidate provided a result similar to that of the enzymatic phosphorylation. In both cases, phosphorylation could not be detected on any amino acid other than histidine. Particularly, no phosphoserine residue was observed.