INOUE Masahoro Institute for Enzyme Research., The University of Tokushima, Assistant Professor, 分子酵素学研究センター, 助手 (00232562)
KIDO Hiroshi Institute for Enzyme Research., The University of Tokushima, Professor, 分子酵素学研究センター, 教授 (50144978)
|Budget Amount *help
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥2,900,000 (Direct Cost: ¥2,900,000)
Hsp70 and 14-3-3 proteins are multifunctional molecular chaperones whose interactions with protein substrates are regulated by ATP hydrolysis and ADP-ATP exchange. In the period granted by this foundation, we found that, in addition to ATPase activity, Hsp70, 14-3-3 proteins and purified 20S proteasome, a new family of N-terminal nucleophile hydrolases, free from nucleoside diphosphate (NDP) kinase, exhibit intrinsic ADP-ATP exchange activity. The rate constants for ATP hydrolysis and ATP synthesis of these proteins were in a similar range at the optimum pH of 7.5-8.5 in the presence of 5 mM ATP and 0.5 mM ADP.During the reaction, these proteins formed acid-labile autophosphorylated intermediates and nucleoside diphosphate-dependent dephosphorylation of the latters then occurred. The 20S proteasome is composed of numerous low molecular mass subunits arranged in a stack of four rings, each containing seven different α- or β-subunits. Among these subunits, we identified that the C5 in the β-type and the C8 in 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. In addition, we recently identified the autophosphorylated amino acid residues, T204 and T211, in Hsp70 and a novel ATP binding site, H227, E231, D232, in HSP70. We are now trying to identify the role of NDP kinase in the chaperone activity of Hsp70 and 14-3-3 proteins and the conformational modification of substrates in the processing of proteolysis by 20S proteasome.