MAGNESIUM AND CATALYTIC RNA 287
atom is positioned approximately 3 Å from the attacked phosphorus atom.
This situation is assigned an inline fi tness value of 1.0. Conversely, for the most
poorly aligned situation (O2 ′ – P - O5 ′ angle for S N 2 nucleophilic attack equals
90 ° in the trigonal bipyramidal transition state), the fi tness parameter has a
value of 0.0. In published crystal structures the position of the 2 ′ - oxygen atom
of the conserved G 8 ribose is located inline with the phosphorus atom and the
5 ′ - oxygen atom of A 9 with the 2 ′ – O – P distance of 2.77 Å. This grouping has
an inline fi tness parameter of 1.17, yet cleavage does not take place at this site.
Scott explains this apparent discrepancy by using a hydrogen - bonding network
among nucleobases at the G 8 , A 9 , and G 12 positions to orbitally steer the neces-
sary lone pair orbitals away from the phosphorus center, thus preventing
nucleophilic attack and bond formation. Scott proposes that the orbital steer-
ing mechanism should be invoked in addition to that of proper alignment of
attacking and leaving groups in the hammerhead cleavage reaction. In pub-
lished crystal structures described up to this time, a considerable conforma-
tional change would be required to achieve a proper inline fi tness parameter
at the cleavage site. For instance, the so - called initial state hammerhead ribo-
zyme structure (PDB: 299D) is in a conformation incompatible with the inline
attack mechanism and has a fi tness parameter of 0.05. In addition to the rate
enhancement that would be achieved by conformational changes toward an
inline arrangement, Scott believes that orbital steering effects could be respon-
sible for some of the 10^6 - fold (compared to the uncatalyzed reaction) rate
enhancement seen for hammerhead cleavage. Other rate enhancement
effects — general acid/base catalysis by metal ions or nucleobases or transition
state stabilization interactions — also are responsible for some cleavage reac-
tion rate enhancement, although orbital steering effects may be involved with
these as well.
Evidence presented in a Journal of Molecular Biology paper of 2002 argued
that a pH - dependent conformational change, rather than the chemical step of
substrate cleavage, could be the rate - limiting step in the hammerhead ribo-
zyme cleavage reaction.^55 First, it is agreed that there is a positive log - linear
relationship between pH and hammerhead cleavage rate up to pH 8.0 (using
Co2+ enhancement) or pH 8.5 (using Mg 2+ enhancement). Also, it is known
that the hammerhead construct used to determine the Scott group crystal
structures (RNA 6 in Figure 6.10 ) contains a kinetic defect that results in a
low concentration of active ribozyme in solution. In solution, the crystal struc-
ture RNA 6 construct exhibits a catalytic rate of 0.08/min in 1.8 M Li 2 SO 4 at
pH 8.5 in the absence or presence of Me 2+ , whereas a kinetically well - behaved
hammerhead exhibits rates of 1.0/min at pH 7.5 in 10 mM MgCl 2. Interestingly,
the cleavage reaction in the crystal obeys the expected log linear relationship
between cleavage rate and pH with a slope of 0.7, the same relationship found
for other hammerhead constructs in solution. Additionally, cleavage in the
crystal displays a biphasic rate dependence with a slow initial pH - dependent
phase and a faster pH - independent phase appearing at pH 6. The faster
rate at or above pH 6 is comparable to the fastest rates observed for other