title: An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects.
abstract: Many of the enzymes that catalyze reactions at nucleotide glycosidic linkages proceed through either a reactive oxocarbenium-ion intermediate or a transition state with considerable oxocarbenium character. To investigate how an RNA active site deals with the catalytic challenge of nucleotide synthesis, we probed the transition state of a ribozyme able to promote the formation of a pyrimidine nucleotide. Primary and secondary kinetic isotope effects indicate that this ribozyme stabilizes a highly dissociative reaction with considerable sp2 hybridization and negligible bond order between the departing pyrophosphate leaving group and the anomeric carbon. The small primary 13C isotope effect of 1.002 +/- 0.003 indicates that the reaction is likely to be less concerted than that observed for protein nucleotide synthesis enzymes, which typically have primary 13C isotope effects of 1.02-1.03. The dissociative nature of the ribozyme reaction most resembles the reaction of some hydrolytic enzymes, such as uracil DNA glycosylase, which uses the negative charges found in the phosphodiester backbone of its DNA substrate to transiently stabilize an oxocarbenium ion during hydrolysis. The detectable hydrolysis observed in the ribozyme reaction indicates that shielding of this reactive intermediate from water is a significant challenge for RNA, which protein enzymes that synthesize nucleotides have managed to overcome during evolution, apparently by the utilization of more concerted chemistry.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6. punrau@sfu.ca
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Unrau
authors.forename: David P
authors.initials: DP
authors.lastname: Bartel
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D006027:Glycosides / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011742:Pyrimidine Nucleotides / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D006027:Glycosides; D011742:Pyrimidine Nucleotides; D016337:RNA, Catalytic