|Budget Amount *help
¥6,000,000 (Direct Cost : ¥6,000,000)
Fiscal Year 1996 : ¥1,600,000 (Direct Cost : ¥1,600,000)
Fiscal Year 1995 : ¥4,400,000 (Direct Cost : ¥4,400,000)
A ribozyme in an RNA enzyme that can catalyze a biochemical reaction. Because a ribozyme requires a metal ions for a catalytic function, the ribozyme is considered as one of the metalloenzymes. Ribozyme reactions are very sensitive to species and concentration of metal ions, suggesting that divalent metal ions may play important roles for the chemical reaction and structural stabilization of the ribozyme.
We have investigated the combined effects of metal ions on both the structural stabilization of the complex between a ribozyme (a leadzyme), CUGGGAGUCC,and a substrate, GGACCGAGCCAG,and the cleavage step in the active center of the complex has been also investigated kinetically.
The results showed that the addition of Nd^<3+> in the presence of Pb^<2+> increased significantly the yield of the RNA cleavage reaction by the leadzyme, although other rare earth ions or divalent ions except Pb^<2+> did not promote the reaction. Further, kinetics for the leadzyme reaction was measured at various concentrations of Nd^<3+> and Pb^<2+>. At low concentration rations of Nd^<3+> under a constant total concentration of metal ions, Nd^<3+> increased the stablility of the complex between the leadzyme and the substrate. In contrast, at high concentration ratios of Nd^<3+>, the addition of Nd^<3+> decreased the stability of the complex. The rate constant of the cleavage step was maximized when the ratio of Nd^<3+> to Pb^<2+> was 1 : 1. These results suggest that the complex between the leadzyme and the substrate has binding sites for Nd^<3+> ion taht influence complex stability and catalyze directly the cleavage reaction. On the basis of the results, we propose a two-metal-ion mechanism in which Pb^<2+> and Nd^<3+> play the roles of base and acid catalyst, respectively.