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 alpha- or beta-subunits. Among the beta-type subunits in the yeast proteasome, three proteolytically active ones were identified, although the functions of the other beta- and alpha-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 gamma-phosphate between NDPs and nucleoside triphosphates. During the transfer of gamma-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 beta-type subunits and C8 among the alpha-type subunits were autophosphorylated during the gamma-phosphate transfer reaction and were photoaffinity labeled with 8-azido-[alpha-(32)P]ATP, suggesting that the C5 and C8 subunits of the proteasome are responsible for the NDP kinase-like activity.
Yano
M
M
Division of Enzyme Chemistry, Institute for Enzyme Research, The University of Tokushima, Tokushima 770, Japan.
Mori
S
S
Kido
H
H
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003565:Cytidine Diphosphate / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D000244:Adenosine Diphosphate; D003565:Cytidine Diphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex