The Structural and Functional Uniqueness of the glmS Ribozyme - Progress in Molecular Biology and Translational Science - page 185

shape of the pH-reactivity curve also suggests that the coenzyme's function
as a general base might precede its function as a general acid. In other words,
maximal activity is provided by deprotonated coenzyme, whose function as
a general base leads to the formation of protonated coenzyme and subse-
quent function as a general acid. Although it has been argued that the appar-
ent role of GlcN6P as a general acid catalyst obviates any invocation of
GlcN6P as a general base catalyst, 13 the rationale presented here makes clear
the necessity that both general base and acid catalysis are somehow inher-
ently interdependent.
A recent study further supports this hypothesis. 41 GlcN6P analogs were
assayed for their ability to support self-cleavage, and a strong correlation was
found between the pH dependence of self-cleavage and the intrinsic acidity
of the GlcN6P analogs. The analogs with low binding affinity exhibited rate
enhancements that were proportional to their intrinsic acidity. This linear
free-energy relationship between coenzyme efficiency and acid dissociation
constant supports a mechanismwherein the coenzyme is directly involved as
a general acid-base catalyst. In this same study, the ligand analogs differed in
their Brønsted acid-base strengths and therefore a Brønsted plot measured
the sensitivity of the self-cleavage reaction to the acid-base strength
of the catalyst. A high value for the Brønsted coefficient was reported
( b

0.7), indicating that the transition state involves a significant amount
of proton transfer. 41
The question remains how the general base catalysis initiated by GlcN6P
at a site distal to the 2 0 -hydroxyl of A-1 can ultimately activate a better-
positioned general base catalyst such as G33. Closer examination of the
proximity of functional groups within the active site of the glmS ribozyme
reveals a plausible mechanism of proton transfer between the coenzyme's
amine functionality and the N1 of G33, as the ultimate general base catalyst
( Fig. 5.4 ). While a proton relay was originally proposed to involve bound
water molecules, 12 which are not observed in other crystal forms of the glmS
ribozyme, 13-15 it has subsequently been proposed 27 that active site nucleo-
tide functional groups proven to be important for catalysis by NAIM support
a scheme for proton transfer ( Fig. 5.4 ). The coenzyme's amine group is spe-
cifically modeled to initiate a proton relay through the intervening N1 of
G32, which contacts a nonbridging phosphate oxygen at the scissile
phosphodiester linkage. In this way, the N1 of G33 and the coenzyme's
amine are simultaneously and necessarily activated to, respectively, serve
as the ultimate general base for deprotonation of the 2 0 -hydroxyl of A-1