The second metal-binding site of 70 kDa heat-shock protein is essential for ADP binding, ATP hydrolysis and ATP synthesis.
The chaperone activity of Hsp70 (70 kDa heat-shock protein) in protein folding and its conformational switch, including oligomeric and monomeric interconversion, are regulated by the hydrolysis of ATP and the ATP-ADP exchange cycle. The crystal structure of human ATPase domain shows two metal-binding sites, the first for ATP binding and a second, in close proximity to the first, whose function remains unknown [Sriram, Osipiuk, Freeman, Morimoto and Joachimiak (1997) Structure 5, 403-414]. In this study, we have characterized the second metal-binding motif by site-directed mutagenesis and the kinetics of ATP and ADP binding, and found that the second metal-binding site, comprising a loop co-ordinated by His-227, Glu-231 and Asp-232, participates both in ATP hydrolysis and ATP-synthetic activities, in co-operation with the first metal-binding site. The first metal-binding site, a catalytic centre, is essential for ATP binding and the second site for ADP binding in the reactions of ATP hydrolysis and ATP synthesis.
Wu
Xueji
X
Division of Enzyme Chemistry, Institute for Enzyme Research, The University of Tokushima, Tokushima 770-8503, Japan.
Yano
Mihiro
M
Washida
Hiroyo
H
Kido
Hiroshi
H
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000096:biosynthesis / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D008670:Metals / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009701:Nucleoside-Diphosphate Kinase / Q000032:analysis / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature
D018840:HSP70 Heat-Shock Proteins; D008670:Metals; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase