ATPase Domain of Hsp70 Exhibits Intrinsic ATP-ADP Exchange Activity

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Y. Mao¹, A. Deng², N. Qu², and X. Wu^2*

¹Medical College and ²School of Life Sciences, Xiamen University-National University of Singapore Laboratory of Biomedical Sciences, Xiamen University, Daxue Road 168, Xiamen 361005, China; fax: +86-592-218-4992; E-mail: xuejiwu@yahoo.com

^* To whom correspondence should be addressed.

Received June 6, 2006; Revision received June 20, 2006

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The chaperone activity of Hsp70 in protein folding and its conformational switching are regulated through the hydrolysis of ATP and the ATP-ADP exchange cycle. It was reported that, in the presence of physiological concentrations of ATP (~5 mM) and ADP (~0.5 mM), Hsp70 catalyzes ATP-ADP exchange through transfer of gamma-phosphate between ATP and ADP, via an autophosphorylated intermediate, whereas it only catalyzes the hydrolysis of ATP in the absence of ADP. To clarify the functional domain of the ATP-ADP exchange activity of Hsp70, we isolated the 44-kD ATPase domain of Hsp70 after limited proteolysis with alpha-chymotrypsin (EC 3.4.21.1). The possibility of ATP-ADP exchange activity of a contaminating nucleoside diphosphate kinase (EC 2.7.4.6) was monitored throughout the experiments. The purified 44-kD ATPase domain exhibited intrinsic ATP-ADP exchange by catalyzing the transfer of gamma-phosphate between ATP and ADP with acid-stable autophosphorylation at Thr204.

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KEY WORDS: ATP-ADP exchange, ATPase domain, autophosphorylation, Hsp70

DOI: 10.1134/S0006297906110071

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