Role of Mg2+ in nucleoside diphosphate kinase autophosphorylation

Nucleoside diphosphate (NDP) kinase is a ubiquitous enzyme that has been described to have regulatory functions, In addition to its classical enzymatic activity, NDP kinases have been characterized as inhibitors of metastasis, as a factor stimulating gene transcription, and as a protein kinase, In this report we show some characteristics of the autophosphorylation of homogeneous NDP kinase and make a comparison with that of other proteins in crude extracts. By using labeled substrates and fluorescence quenching analysis, we prove that Mg2+ is indeed necessary for the two steps of the ping-pong reaction to take place and present evidence that NTPs or NDPs, when uncomplexed to divalent cations, may not bind the active site in a comparable way to NTP Mg2+ and NDP Mg2+, However, even extremely small concentrations of Mg2+ suffice for maximal autophosphorylation which is obtained with Mg2+ in the nanomolar range and 100 mu M ATP using homogeneous enzyme. Moreover, lower autophosphorylation levels were observed with increasing concentrations of Mg2+. The autophosphorylation equilibrium varied from 0.19 to 1.6 upon the inclusion of 10 mM EDTA to produce low Mg2+ concentrations. Under optimal conditions (low Mg2+ concentrations and short incubation times) NDP kinase was the only protein phosphorylated in crude extracts from Candida albicans, indicating that the autophosphorylation properties of the enzyme are very singular. (C) 1998 Academic Press.