Bridging the Gap Between Theory and Experiment to Derive a Detailed Understanding of Hammerhead Ribozyme Catalysis - Progress in Molecular Biology and Translational Science - page 60

activated precursor state, this is accomplished by an Mg 2 þ ion and an addi-
tional Na þ ion, or three Na þ ions. Moreover, to form active in-line con-
formations, these ions must adopt specific bridging coordination patterns:
either a bridging Mg 2 þ ion or specific patterns of bridging Na þ ions.
It has been well established that, in the absence of divalent ions, the
HHR retains activity at high concentration of monovalent ions. 131 The
properties of HHR cleavage in high concentrations of monovalent ions
are similar to those in the presence of divalent metal ions, which have been
interpreted to indicate that the major role of the cations is simple electro-
static stabilization of the phosphates to allow folding into an active confor-
mation. However, there remains some smaller contribution to the rate
enhancement that can be effected through a more active role played by at
least one divalent metal ion, as observed by the reduction in rate in the pres-
ence of only monovalent ions 130 or exchange inert ions. 132
There are notable exceptions whereby HHR cleavage differed signif-
icantly in the presence and absence of divalent metal ions. Disruption of
implicated divalent metal ion-binding sites at G10.1:N7, A9:O2P, and
C1.1:O2P has significant deleterious effects on HHR cleavage in the pres-
ence of divalent ions, but not in high concentrations of Li þ .Forexample,
both A9:O2P and C1.1:O2P exhibit significant catalytic thio effects that
can be rescued by thiophilic ions such as Cd 2 þ .Moreover,bothG10.1:
N7 and A9:O2P form a divalent metal ion-binding site as pinpointed by
electron spin-echo envelope modulation 138 and as observed crystallo-
graphically for the eHHR. 74 The present simulation results are consistent
with experimental evidence and indicate that threshold occupancy and
specific coordination patterns of either Mg 2 þ or Na þ can electrostatically
stabilize the active site and facilitate active in-line conformations. These
results provide detailed insight into the specific roles played by divalent
and monovalent ions.
It has been demonstrated that the sensitivity of HHR activity to divalent
ions is reduced upon introduction of tertiary stabilizing motifs. 121,139-142
A recent study of the tertiary stabilized RzB HHR has led to the suggestion
that HHR catalysis may occur through a multichannel mechanism that has
available both a divalent-dependent and divalent-independent pathways. 121
These experiments, together with previous measurement of pH-rate
profiles, 141 are consistent with the interpretation that the divalent ion
may play a specific nonstructural role in catalysis. The present simulation
results provide insight into the nature of the different metal ion-binding pat-
terns that give rise to catalytically active conformations, and further support