Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.
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 ATPgammaS. Identical ionic conditions yield a thiophosphorylated strand when ATPgammaS is included, thus effecting a net ATPgammaS 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 ATPgammaS 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.
Saran
Dayal
D
Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.
Held
Daniel M
DM
Burke
Donald H
DH
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
C506620:2PTmin3.2 ribozyme; D008670:Metals; D016337:RNA, Catalytic; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); D006867:Hydrolases; D000251:Adenosine Triphosphatases