BIOINORGANIC CHEMISTRY A Short Course Second Edition

278 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II

displaces a Mg 2+ ion that is essential for catalysis. The fi rst hypothesis asserts
that Mg 2+ assists in forming a transition state structure, not that observed in
the crystal structure, that maintains a particular orientation between G 5 and
nearby stem (helix) III. The second asserts that (a) Mg 2+ must be released
before G 5 can participate in a transition state structure and (b) Tb(III) with
its tighter binding is not released, thereby blocking the required conforma-
tional change. The authors tested the hypotheses by placing a bulky group at
the 2 ′ - hydroxyl position of U 15.3 in stem (helix) III fi nding that the Tb(III)
inhibition (and presumably Tb(III) binding) did not take place but catalytic
cleavage took place in the presence of Mg 2+. This experiment appears to refute
the fi rst hypothesis above — Mg 2+ assists in forming a transition state structure
that maintains a particular orientation between G 5 and nearby stem (helix)
III — while leaving the second hypothesis viable and consistent with mechanis-
tic data available in 1998 — the Mg 2+ binding to G 5 must be released before G 5
can participate in a transition state structure. However, the actual role of G 5
in hammerhead ribozyme catalysis remained open and in need of further
experimentation.
Scott ’ s group continued its study of hammerhead X - ray crystallographic
structure with the aim of understanding the conformational change required
for ribozyme self - cleavage.^44 To create a kinetic bottleneck that would prevent
the fi nal chemical cleavage reaction step in the crystal, the researchers intro-
duced a cleavage site modifi cation — a talo - 5 ′ - C - methyl modifi ed ribose on A 1.1
at the C17 - A1.1 cleavage site (see Figure 6.17 ).
Through this modifi cation, they hoped to capture a later conformational
intermediate poised to form an inline transition state. The technique used was
that of time - resolved crystallography, one that has successfully captured
protein intermediates (see Section 3.7.2.3). The data determining the crystal
structure has been deposited in the Protein Databank as PDB: 379D.

Figure 6.17 Talo - 5 ′ C - methyl substituent at the hammerhead ribozyme active site.

C17

HO O

O

O

O

• O PO
  OH

CH 3

H

3'

2'

5'

A1.1 talo-5' C-methyl substituent