Phosphorylation mechanism of nucleoside diphosphate kinase: 31P-nuclear magnetic resonance studies.
The phosphorylation mechanism of Dictyostelium discoideum nucleoside diphosphate (NDP) kinase was investigated by NMR. 31P 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.
Lecroisey
A
A
Laboratoire de Résonance Magnétique Nucléaire, CNRS URA 1129, Institut Pasteur, Paris, France.
Lascu
I
I
Bominaar
A
A
Véron
M
M
Delepierre
M
M
D000818:Animals; D001665:Binding Sites; D004023:Dictyostelium / Q000201:enzymology; D006639:Histidine / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010759:Phosphorus Isotopes; D010766:Phosphorylation; D015800:Protozoan Proteins / Q000378:metabolism
D010750:Phosphoproteins; D010759:Phosphorus Isotopes; D015800:Protozoan Proteins; D006639:Histidine; D009701:Nucleoside-Diphosphate Kinase