Published online 21 June 2006
© 2006 The Author(s)
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Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme
1 Department of Chemistry, Indiana University Bloomington, IN 47405, USA 2 Department of Biology, Indiana University Bloomington, IN 47405, USA 3 Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine Columbia, MO 65211, USA 4 Department of Biochemistry, University of Missouri School of Medicine Columbia, MO 65211, USA
*To whom correspondence should be address. Tel: +1 573 884 1316; Fax: +1 573 884 9676; Email: burkedh{at}missouri.edu
Received March 1, 2006. Revised April 25, 2006. Accepted May 19, 2006.
Ribozymes that phosphorylate internal 2'-OH positions mimic the first mechanistic step of P-type ATPase enzymes by forming a phospho-enzyme intermediate. We previously described 2'-autophosphorylation and autothiophosphorylation by the 2PTmin3.2 ribozyme. In the present work we demonstrate that the thiophosphorylated form of this ribozyme can de-thiophosphorylate in the absence of ATP
S. Identical ionic conditions yield a thiophosphorylated strand when ATPS is included, thus effecting a net ATPS hydrolysis. The de-thiophosphorylation step is nearly independent of pH over the range of 6.3–8.5 and does not require a specifically folded RNA structure, but this step is greatly stimulated by transition metal ions. By monitoring thiophosphate release, we observe 29–46 ATPS hydrolyzed per ribozyme strand in 24 h, corresponding to a turnover rate of 1.2–2.0 h–1. The existence of an ATP- (or thio-ATP-)powered catalytic cycle raises the possibility of using ribozymes to transduce chemical energy into mechanical work for nucleic acid nanodevices.
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