The Structural and Functional Uniqueness of the glmS Ribozyme - Progress in Molecular Biology and Translational Science - page 177

is open and solvent-accessible. 12 Finally, crystallographic data have aided in
defining contacts between RNA functional groups and the ligand sugar
ring. 12-15,17 As additional crystal structures have been solved, more insight
has been gained into the active site and coenzyme-binding site. Helix P2.2
and the G1 nucleotide within the active glmS ribozyme have been shown to
be key features necessary for coenzyme binding and subsequent catalysis. 15
In addition, X-ray crystallography and kinetic experiments have identi-
fied a site-specific mutation within the catalytic core that results in a loss of
catalysis but not coenzyme binding, indicating that a conserved guanine
(G33) within the active site plays a role in the catalytic mechanism. 14,17 Fur-
thermore, crystallographic studies with the competitive inhibitor Glc6P and
the nonnatural sugar mannosamine-6-phosphate (MaN6P) revealed similar
binding interactions with the RNA as observed for the natural ligand. Glc6P
cannot support glmS self-cleavage, but MaN6P does at a rate about sevenfold
slower than GlcN6P. 6,17 However, the binding affinity for MaN6P is wea-
ker compared to GlcN6P, which is likely an important indication of how the
ribozyme selects against ligands that are structurally and chemically similar to
GlcN6P. 17

7. METAL ION USAGE BY THE glmS RIBOZYME
For many ribozymes, metal ion binding is a key feature of RNA struc-
ture and/or function. While all ribozymes require metal ions for folding, few
utilize essential metal ions in their catalytic mechanisms. Therefore, many
ribozymes retain catalytic activity, although sometimes at a reduced rate,
as long as the folding requirements are fulfilled by monovalent, divalent,
or multivalent metal ions. Although physiologically divalent metal ions
are probably most relevant to glmS structure and function, self-cleavage
activity is supported by an exchange-inert cobalt(III) complex and by molar
concentrations of monovalent ions. 30,31 Cobalt hexamine is a structural
mimic of hexahydrated Mg 2 þ and is inert because of exchange of inner
sphere amines and protons being slow relative to the self-cleavage reaction.
Therefore, interaction of cobalt hexamine with RNA can only occur
through electrostatic and outer sphere interactions. 32,33 glmS self-cleavage
is supported by cobalt hexamine (at 1 mM) at a rate only slightly slower

than that obtained in the presence of saturating Mg 2 þ , indicating that the
ribozyme does not require inner sphere divalent metal ion coordination
for catalysis.