American Chemical Society
ja207426j_si_001.pdf (2.84 MB)

An Active-Site Guanine Participates in glmS Ribozyme Catalysis in Its Protonated State

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journal contribution
posted on 2011-11-16, 00:00 authored by Júlia Viladoms, Lincoln G. Scott, Martha J. Fedor
Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2′-OH nucleophile. Apparent pKa values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pKa values obtained from functional assays and microscopic pKa values obtained from pH–fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.