Intrinsic Nucleoside Diphosphate Kinase-like Activity Is a Novel Function of the 20 S Proteasome

The eukaryotic 20 S proteasome is the prototype of a new family of the N-terminal nucleophil hydrolases and is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different α- or β-subunits. Among the β-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other β- and α-type subunits remain to be clarified. We report here that the purified 20 S proteasome exhibits intrinsic nucleoside diphosphate (NDP) kinase-like activity. The proteasome exhibited a preference for ATP and dATP as phosphate donors, and a broad specificity for NDPs, other than GDP, as phosphate acceptors, unlike conventional NDP kinase, which catalyzes the transfer of γ-phosphate between NDPs and nucleoside triphosphates. During the transfer of γ-phosphate, the proteasome formed acid-labile phosphohistidine as autophosphorylated intermediates, and NDP-dependent dephosphorylation of the latter then occurred. These enzymatic properties are similar to those of the molecular chaperone, Hsp70, which also exhibits intrinsic NDP kinase-like activity, instead of ATPase activity. C5 among the β-type subunits and C8 among the α-type subunits were autophosphorylated during the γ-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[α- 32 P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.