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Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.

Saran D, Held DM, Burke DH - Nucleic Acids Res. (2006)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.

ABSTRACT
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.

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Related in: MedlinePlus

Temperature and pH dependence of de-thiophosphorylation reaction. (A) De-thiophosphorylation of substrate strand DRD26 was monitored at 20°C (asterisk), 30°C (circle), 40°C (diamond), 50°C (triangle) and 55°C (square). (B) Fraction de-thiophosphorylated at 2 h (circles) or 4 h (diamonds) is plotted as a function of the temperature of the reaction, or (C) as a function of pH. Reactions carried out at pH 6.4, 7.1 and 7.6 used 50 mM PIPES as the pH buffer; those carried out at pH 7.7, 8.1 and 8.5 used 50 mM Tris–HCl.
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fig3: Temperature and pH dependence of de-thiophosphorylation reaction. (A) De-thiophosphorylation of substrate strand DRD26 was monitored at 20°C (asterisk), 30°C (circle), 40°C (diamond), 50°C (triangle) and 55°C (square). (B) Fraction de-thiophosphorylated at 2 h (circles) or 4 h (diamonds) is plotted as a function of the temperature of the reaction, or (C) as a function of pH. Reactions carried out at pH 6.4, 7.1 and 7.6 used 50 mM PIPES as the pH buffer; those carried out at pH 7.7, 8.1 and 8.5 used 50 mM Tris–HCl.

Mentions: When DRD26 de-thiophosphorylation kinetics were monitored over the temperature range of 20–55°C, the amount of de-thiophosphorylation after 4 h increased with increasing temperature (Figure 3A). Some of this effect may be due to enhanced intrinsic chemical reactivity with increasing temperature. However, the magnitude of the de-thiophosphorylation increased markedly between 30 and 40°C (Figure 3B). This apparently cooperative transition supports a model in which inhibitory secondary structures melt over this range to expose the 2′-thiophosphate to hydrolysis. The shortening of the lag phase of the reaction at elevated temperatures is especially evident when yield after 2 h is compared across the temperature range (Figure 3B, circles).


Multiple-turnover thio-ATP hydrolase and phospho-enzyme intermediate formation activities catalyzed by an RNA enzyme.

Saran D, Held DM, Burke DH - Nucleic Acids Res. (2006)

Temperature and pH dependence of de-thiophosphorylation reaction. (A) De-thiophosphorylation of substrate strand DRD26 was monitored at 20°C (asterisk), 30°C (circle), 40°C (diamond), 50°C (triangle) and 55°C (square). (B) Fraction de-thiophosphorylated at 2 h (circles) or 4 h (diamonds) is plotted as a function of the temperature of the reaction, or (C) as a function of pH. Reactions carried out at pH 6.4, 7.1 and 7.6 used 50 mM PIPES as the pH buffer; those carried out at pH 7.7, 8.1 and 8.5 used 50 mM Tris–HCl.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1484251&req=5

fig3: Temperature and pH dependence of de-thiophosphorylation reaction. (A) De-thiophosphorylation of substrate strand DRD26 was monitored at 20°C (asterisk), 30°C (circle), 40°C (diamond), 50°C (triangle) and 55°C (square). (B) Fraction de-thiophosphorylated at 2 h (circles) or 4 h (diamonds) is plotted as a function of the temperature of the reaction, or (C) as a function of pH. Reactions carried out at pH 6.4, 7.1 and 7.6 used 50 mM PIPES as the pH buffer; those carried out at pH 7.7, 8.1 and 8.5 used 50 mM Tris–HCl.
Mentions: When DRD26 de-thiophosphorylation kinetics were monitored over the temperature range of 20–55°C, the amount of de-thiophosphorylation after 4 h increased with increasing temperature (Figure 3A). Some of this effect may be due to enhanced intrinsic chemical reactivity with increasing temperature. However, the magnitude of the de-thiophosphorylation increased markedly between 30 and 40°C (Figure 3B). This apparently cooperative transition supports a model in which inhibitory secondary structures melt over this range to expose the 2′-thiophosphate to hydrolysis. The shortening of the lag phase of the reaction at elevated temperatures is especially evident when yield after 2 h is compared across the temperature range (Figure 3B, circles).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Indiana University, Bloomington, IN 47405, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus