Biology Reference
In-Depth Information
An important feature of the natural ligand GlcN6P is the phosphate
group, which increases the p
K
a
of the amine functionality in solution.
Although GlcN can stimulate ribozyme self-cleavage, the reaction rate
and apparent affinity were substantially reduced, reflecting the contribution
of phosphate interactions with the RNA and metal ion upon binding.
6
Fur-
thermore, the solution p
K
a
for GlcN was essentially equal to the apparent
p
K
a
for
glmS
ribozyme self-cleavage with GlcN or GlcN6P.
6
These data
suggested that ligand phosphate influence on p
K
a
is quenched upon inter-
action with the ribozyme and substantially shifted toward neutrality. Fur-
thermore, glucosamine-6-sulphate can support ribozyme self-cleavage to
the same extent as GlcN6P, albeit at concentrations at least 100-fold greater.
The phosphate group therefore plays a role in recognition of the ligand by
the RNA and effects protonation state of
the coenzyme relevant
to
catalysis.
6,12-14,17-19,27,31,36-42
Further synthesis and functional analysis of ligand analogs will elucidate
the intricacies of ligand binding and its role as the coenzyme for
glmS
self-
cleavage. Although many specific interactions occur between the
glmS
RNA and GlcN6P allowing for discrimination against closely related com-
pounds, there appears to remain some flexibility for design of novel ligand
analogs that either support or inhibit
glmS
self-cleavage. Such compounds
might work as agonists or antagonists to perturb expression of the GlmS
enzyme and amino sugar metabolism.
9. MECHANISM OF glmS RIBOZYME SELF-CLEAVAGE
USING COENZYME GlcN6P
The role of ligand as a coenzyme has been described; however, its
exact function in the mechanism of self-cleavage is still under investigation.
A number of hypotheses regarding the identity of the general acid and base
functional groups for
glmS
self-cleavage have come from different biochem-
ical and biophysical analyses.
glmS
ribozyme structure appears unperturbed
by GlcN6P binding,
28
and self-cleavage cannot occur in the absence of
GlcN6P or related amine-containing compounds,
6
suggesting the metabo-
lite is involved in the mechanism of catalysis. Moreover, the role of ligand as
coenzyme was first supported by evidence that the apparent reaction p
K
a
for
catalysis corresponds to the p
K
a
of the amine functionality of GlcN6P and
other analogs.
6
The pH dependence of
glmS
self-cleavage implies that
GlcN6P acts as the general acid and/or base for the reaction. The role of
GlcN6P as a coenzyme was further validated by crystallography, which
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