296 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
could constitute a biochemical switch that might control whether the RNA
functions as a nuclease (cleavage) or a ligase (reversal of the cleavage
reaction).
In 2005, Kenneth Blount and Olke Uhlenbeck published an extensive
review of biochemical, spectroscopic, and structural research results obtained
for the hammerhead ribozyme up to that date.^61 The review is aptly titled “ The
Structure – Function Dilemma of the Hammerhead Ribozyme. ” In the abstract
to this article the authors state that the structural and functional data obtained
so far for the hammerhead ribozyme do not agree well and that these disagree-
ments have prevented a unifi ed catalytic mechanism from emerging. In the
article, they defi ne a “ consensus set of functional groups unambiguously
required for hammerhead catalysis. ” Looking at this set of functional groups
in relation to the available crystal structures helped the reference 61 authors
defi ne a concise set of disagreements between structural and functional data.
First, these authors describe the natural hammerhead ribozyme as a uni-
molecular species containing helices I, II, and III. Helices I and II are closed
by loops. The constructs used in in vitro experiments as described in the above
discussion are all bimolecular species. The “ substrate ” strand contains the
scissile phosphate, while the other strand is called the “ ribozyme. ” A typical
hammerhead contains a 15 - residue conserved catalytic core domain — adjacent
to the cleavage site between residues 17 and 1.1 on the substrate strand —
consisting of residues C 3 , U 4 , G 5 , A 6 , and U 7 (not conserved) and G 8 , A 9 , G 10.1 ,
C11.1 , G 12 , A 13 , A 14 , A 15 , U 16.1 , and C 17 (not conserved). Hammerhead catalysis is
described by three steps in a manner similar to that for protein enzymes: (1)
association of ribozyme and substrate; (2) the chemical cleavage step; and (3)
dissociation of the ribozyme – products complex. The chemical cleavage step, a
transesterifi cation, is uniform for all hammerheads. The reverse reaction, a
ligation, is also catalyzed but at a 100 - fold lower rate. The chemical cleavage
step takes place when the 2 ′ OH of substrate residue 17.0 is deprotonated and
attacks the phosphorus of residue 1.1, forming a fi ve - coordinate trigonal bipy-
ramidal transition state. The 3 ′ - product consists of the nucleotide 1.1 ribose ’ s
5 ′ - oxygen as the leaving group that is expelled and subsequently protonated.
The 5 ′ - product of the cleavage is a 2 ′ ,3 ′ - cyclic phosphate (see Figure 6.9 ).
Because the chirality of the scissile phosphate is inverted over the course of
the reaction, an S N 2(P) mechanism is implied in which the 2 ′ - oxygen nucleo-
phile must be in line with the scissile phosphorus - 5 ′ - oxygen bond for cleavage
to occur. Hammerhead cleavage may be catalyzed through (1) general base
catalysis — deprotonation of the 2 ′ - OH; (2) general acid catalysis — protonation
of the 5 ′ - O leaving group; and (3) transition state stabilization — possibly by
negative charge reduction through interaction with cations. Magnesium ions —
for example, coordinated near the cleavage site — are proposed to fi ll this role
in catalysis enhancement.
Using an approach that has been successful with protein enzymes and other
ribozymes, researchers have used hammerhead X - ray crystallographic struc-
tures to identify sites around the catalytic center where mutation might yield