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Volume 272, Number 43, Issue of October 24, 1997 pp. 26822-26826
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Involvement of a Specific Metal Ion in the Transition of the Hammerhead Ribozyme to Its Catalytic Conformation

(Received for publication, July 14, 1997, and in revised form, August 27, 1997)

Alessio Peracchi , Leonid Beigelman ^¶ , Edmund C. Scott , Olke C. Uhlenbeck and Daniel Herschlag

From the  Department of Biochemistry, Stanford University, Stanford, California 94305-5307, ^¶ Ribozyme Pharmaceuticals Inc., Boulder, Colorado 80301, and the  Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-215

Previous crystallographic and biochemical studies of the hammerhead ribozyme suggest that a metal ion is ligated by the pro-R_p oxygen of phosphate 9 and by N₇ of G10.1 and has a functional role in the cleavage reaction. We have tested this model by examining the cleavage properties of a hammerhead containing a unique phosphorothioate at position 9. The R_p-, but not S_p-, phosphorothioate reduces the cleavage rate by 10³-fold, and the rate can be fully restored by addition of low concentrations of Cd²⁺, a thiophilic metal ion. These results strongly suggest that this bound metal ion is critical for catalysis, despite its location ~20 Å from the cleavage site in the crystal structure. Analysis of the concentration dependence suggests that Cd²⁺ binds with a K_d of 25 µM in the ground state and a K_d of 2.5 nM in the transition state. The much stronger transition state binding suggests that the P9 metal ion adopts at least one additional ligand in the transition state and that this metal ion may participate in a large scale conformational change that precedes hammerhead cleavage.

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