The Structural and Functional Uniqueness of the glmS Ribozyme - Progress in Molecular Biology and Translational Science - page 186

and the general acid for protonation of the 5 0 -oxygen leaving group of G1.
Importantly, NAIM experiments demonstrated the dependence of glmS
ribozyme activity on the 2 0 -hydroxyl groups of U59 and A58. 27 The
U59 residue may influence the p K a of G33 through interaction at its N3
position, while A58 promotes or stabilizes formation of the 2 0 -oxygen
nucleophile at the scissile phosphodiester linkage. A recent study examined
the pH dependence of self-cleavage by wild type and mutant glmS ribozymes
and found that the apparent p K a values did not support a role for G33 or any
other active site guanine in general base catalysis. Further work using
pH-fluorescence profiles with ribozymes containing a fluorescent guanosine
analog, 8-azaguanosine, at position 33 suggested that the pH-dependent step
in catalysis did not involve G33 deprotonation. 40 These results are
strengthened by molecular dynamics work that determined that G40
( T. tengcongensis ribozyme numbering corresponding to G33 in the
B. cereus ribozyme) played a structural stabilization role within the active site
but not a direct chemical role. 37 These results point to an alternative role for
G40/G33, specifically for the protonated N1 position, in proton transfer
and/or transition state stabilization. 40
The nucleobase identity at G32 is strictly conserved in glmS
ribozymes 9,35 and preliminary results indicate that mutation at this site results
in substantial loss of self-cleavage activity (J. Soukup, unpublished). The
proposed comprehensive model ( Fig. 5.4 ) appropriately predicts that any
perturbation in the chain of events in the proton relay is equally detrimental
to ribozyme activity (i.e., general base and acid catalysis are inherently
interdependent). Therefore, the proposed mechanism is consistent with
the entirety of available biochemical and biophysical data.
11. POTENTIAL FOR ANTIBIOTIC DEVELOPMENT
AFFECTING glmS RIBOZYME/RIBOSWITCH
FUNCTION
Biochemical and biophysical analyses have provided considerable
information regarding the unique coenzyme-dependent catalysis of the glmS
ribozyme. Coenzyme functional groups required for binding, and the coen-
zyme amine functionality and p K a perturbation are all essential aspects that
impact optimal glmS ribozyme self-cleavage activity. Although the require-
ments for efficient ligand binding and catalysis are many, there appear to
remain opportunities for development of compounds that effect ribozyme
self-cleavage and riboswitch function. In bacteria, the riboswitch must be