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(Received for publication, July 14, 1997, and in revised form, August 27, 1997)
From the Previous crystallographic and biochemical studies
of the hammerhead ribozyme suggest that a metal ion is ligated by the
pro-Rp 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 Rp-, but not
Sp-, 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 Kd of
25 µM in the ground state and a Kd 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.
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
,
,
and
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
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