The Hammerhead Ribozyme: Structure, Catalysis, and Gene Regulation - Progress in Molecular Biology and Translational Science - page 7

was not apparent from the crystal structure of the minimal hammer-
head 13-15,44 what functional groups might be involved in acid-base catalysis.
Consequently, the focus of biochemical mechanistic investigations in the
hammerhead turned to this problem.
The invariant core residues G12 and G8 in the hammerhead ribozyme
were finally identified in 2005 as likely candidates for participation in acid-
base chemistry by careful purine modification studies conducted by John
Burke and coworkers. 45,46 Substitution of G12 (p K a 9.5) with inosine
(p K a 8.7), 2,6-diaminopurine (p K a 5.1) or 2-aminopurine (p K a 3.8) shifts
the reaction rate profile in a manner consistent with G12's suggested role
in general base (or acid) catalysis without significantly perturbing ribozyme
folding. 45 Similar substitutions at G8 also implicated this invariant residue in
acid-base catalysis, but in this case, the modifications also partially inhibited
ribozyme folding. 45 These experiments could not determine specifically
whether an individual nucleotide, such as G12, was the general acid or
the general base, but clearly implicated G12 and G8 in acid-base catalysis.

2.1.4 Kinetics
The minimal hammerhead ribozyme, under “standard” reaction conditions
(10 mM Tris, pH 7.5, 10 mM MgCl 2 ) has a turnover rate on the order of
1 min 1 ,a K m of about 10 m m, and a log-linear dependence of rate on
pH with a slope of 0.7. Above pH 8.5-9.0 (depending upon reaction con-
ditions), the rate becomes pH independent, suggesting an apparent kinetic
p K a of about 8.5-9.0. 25,47,48 This observation is consistent with both Mg 2 þ -
and guanine-mediated acid-base chemistry. The full-length hammerhead
ribozyme shows similar pH dependence, but the cleavage rate is up to
1000-fold enhanced (i.e., approximately 15 s 1 ). 49 There exists no compel-
ling evidence that the reaction is sequential rather than concerted, although
this remains an issue for debate. It is perplexing that the pH dependence of
the rate-limiting step is similar in both the minimal and full-length
ribozymes, despite the remarkable reaction rate difference.

2.2. Crystal structure
The crystal structure of a minimal hammerhead ribozyme ( Fig. 1.1 B) was
the first near-atomic resolution structure of a ribozyme to be deter-
mined. 13-15 However, the minimal hammerhead ribozyme sequence crys-
tallizes in what is now recognized as an “open,” apparently precatalytic
conformation 50,51 in which four of the invariant residues (C3, U4, G5,
and A6) form a uridine turn structure 13,52

similar to that found in the