286 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
(A 6 N 6 – O 1P = 2.7 Å ) and (2) the close approach of G 5 ’ s keto oxygen (O 6 ) and
the 2 ′ - oxygen of C 17 (G 5 O 6 – 2 ′ - O = 2.9 Å ). These interactions are most interest-
ing because they involve groups on G 5 and A 6 known to be required for ham-
merhead catalytic cleavage. The authors speculate that the 2 ′ - OH deprotonation
necessary to form the 2 ′ ,3 ′ - cyclic phosphate product might result in a transient
G 5 enol form that would produce a G 5 O 6 – 2 ′ - O hydrogen bond as described in
(2) above. Additionally, C 17 appears to form a perpendicular stabilizing aro-
matic interaction with G 5 similar to those found for aromatic substituents in
protein structures. The visualized 2 ′ ,3 ′ - cyclic phosphate resembles a pentaco-
ordinated 2 ′ ,3 ′ ,5 ′ - cyclic oxyphosphorane transition state; however, the 5 ′ - O
connection appears to be missing in the visualized crystal structure. Therefore,
the issue of 5 ′ - oxygen stabilization during cleavage is not addressed in this
structure, leaving the authors to hope for a stable transition state analog that
mimics a 5 - coordinate oxyphosphorane intermediate in future research.
In an earlier X - ray crystallographic study the Scott research group found
that terbium(III) inhibited the hammerhead cleavage reaction (reference 43 ,
PDB: 359D) and also found a Tb 3+ ion bound to the Watson – Crick face of G 5.
In that work, the authors proposed that this Tb 3+ prevented G 5 from making
the interactions required for catalysis. This fi nding agrees with the importance
of C 17 – G 5 and A 6 interactions found in the PDB: 488D structure. Research by
other groups has confi rmed that the exocyclic groups of both A 6 and G 5 are
required for competent catalytic cleavage in the hammerhead ribozyme. The
2000 Molecular Cell paper does not address changes in domain II (augmented
stem II), especially those discussed previously in the A 9 /G 10.1 region, and does
not provide any insight as to the catalytic role of conserved nucleotides in that
region.
In summary, the reference 52 authors propose the following: (1) A confor-
mational rearrangement of the cleavage site nucleotide C 17 positions the
attacking 2 ′ - O nucleophile in line with the 5 ′ - O leaving group, allowing the
SN 2 reaction to proceed (see Figure 6.9 ); (2) interactions between the scissile
phosphate and ribose of C 17 and exocyclic functional groups of G 5 and A 6 are
involved in establishing the active intermediate; and (3) the RNA itself may
possess the necessary catalytic properties and may not require divalent metal
ions for the cleavage reaction. Their results do not (1) address how the 5 ′ -
oxygen leaving group is stabilized, (2) explain the critical requirement for C 3
in hammerhead catalytic cleavage, or (3) rule out the proposed Herschlag
mechanism in which the A 9 phosphate and scissile phosphate coordinate a
single metal ion (but see objections in reference 49 , discussed above).
In 2001, Scott published a paper that discussed ribozyme catalysis in terms
of “ orbital steering ” and “ inline fi tness. ”^53 In defi ning the former process, it has
been suggested that the catalytic effi ciency of enzymes depends on their ability
not only to juxtapose the reacting atoms but also to “ steer ” their orbitals along
a path that takes advantage of this strong directional preference.^54 In the latter,
an inline fi tness parameter has been devised in which a perfectly aligned
(O2′ – P – O5 ′ angle for S N 2 nucleophilic attack equals 180 ° ), and the 2 ′ - oxygen