title: An active-site guanine participates in glmS ribozyme catalysis in its protonated state.
abstract: 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 pK(a) 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 pK(a) values obtained from functional assays and microscopic pK(a) 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.
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
authors.forename: Júlia
authors.initials: J
authors.lastname: Viladoms
authors.forename: Lincoln G
authors.initials: LG
authors.lastname: Scott
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Fedor
mesh_terms: D001408:Bacillus anthracis / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D006147:Guanine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016337:RNA, Catalytic; D006147:Guanine