An oxocarbenium-ion intermediate of a ribozyme reaction indicated by kinetic isotope effects Peter J. Unrau†‡ and David P. Bartel‡§ †Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6; and §Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, 9 Cambridge Center, Cambridge, MA 02142 Edited by Thomas R. Cech, Howard Hughes Medical Institute, Chevy Chase, MD, and approved October 14, 2003 (received for review May 23, 2003) Many of the enzymes that catalyze reactions at nucleotide glyco- take place through a very weakly concerted oxocarbenium-like sidic linkages proceed through either a reactive oxocarbenium-ion transition state (5), a ribozyme, in analogy with protein enzymes, intermediate or a transition state with considerable oxocarbenium could employ at least two distinct catalytic strategies. One would be character. To investigate how an RNA active site deals with the to promote the oxocarbenium-ion character of the uncatalyzed catalytic challenge of nucleotide synthesis, we probed the transi- transition state while simultaneously limiting access of water to this tion state of a ribozyme able to promote the formation of a reactive state (Fig. 1a). Alternatively, a ribozyme might promote a pyrimidine nucleotide. Primary and secondary kinetic isotope ef- concerted mechanism in which the attack of the 4SUra and the fects indicate that this ribozyme stabilizes a highly dissociative departure of the pyrophosphate are made more synchronous reaction with considerable sp2 hybridization and negligible bond (Fig. 1b). The relative accessibility of these catalytic strategies for order between the departing pyrophosphate leaving group and emergent ribozymes is difficult to assess based on previous studies, the anomeric carbon. The small primary 13C isotope effect of because prior exploration of ribozyme reaction mechanisms and -indicates that the reaction is likely to be less catalytic strategies has focused on ribozymes with concerted reac 0.003 ؎ 1.002 concerted than that observed for protein nucleotide synthesis tions (13, 14). enzymes, which typically have primary 13C isotope effects of Examining the effects of isotopic substitution at or near the 1.02–1.03. The dissociative nature of the ribozyme reaction most substrate reactive center has provided an invaluable tool for char- resembles the reaction of some hydrolytic enzymes, such as uracil acterizing the transition states of proteinaceous enzymes (15, 16). DNA glycosylase, which uses the negative charges found in the Isotopic modification of an enzyme substrate leaves the static phosphodiester backbone of its DNA substrate to transiently sta- (geometric) arrangement of atoms unchanged but modifies the bilize an oxocarbenium ion during hydrolysis. The detectable kinetic (vibrational and translational) properties of the transition hydrolysis observed in the ribozyme reaction indicates that shield- state and thus provides an experimental avenue for examining ing of this reactive intermediate from water is a significant chal- transition-state structure. To extend the analysis of kinetic isotope lenge for RNA, which protein enzymes that synthesize nucleotides effects (KIEs) to the study of a ribozyme transition state, we have managed to overcome during evolution, apparently by the synthesized ribozyme-PRPP molecules with isotopic substitutions utilization of more concerted chemistry. of key PRPP atoms and compared the ribozyme reaction rates of the substituted molecules with those of analogously prepared e previously isolated from a large pool of random RNA molecules with natural isotopic abundance. Wsequences a ribozyme able to synthesize a pyrimidine Materials and Methods nucleotide in a reaction modeled after pyrimidine synthesis of Preparation of Radiolabeled Ribozyme-PRPP Pairs. contemporary metabolism (1, 2). The ribozyme promotes the In vitro evolution formation of 4-thiouridine at its 3Ј terminus, using tethered was used to generate a variant of isolate a15 from our initial selection with an efficiency suitable for characterization by using 5-phosphoribosyl 1-pyrophosphate (PRPP) and free 4-thiouracil Ј kinetic isotope methods (1, 2) (improved sequence: 5 -GGA BIOCHEMISTRY (4SUra). This ribozyme and its catalytic mechanism are of UAA UGC GCA CAA GUA AGC GUG UUG CAA UCC interest because pyrimidine nucleotide synthesis is a challenging UGC CUA UUG AAA CAC GAA AUC AGU CGU AAU reaction, central to the RNA world, that has not been demon- GGA AAC GAG GGG GAU AGG UUG CUC GUU ACU strated by prebiotic chemistry (3, 4). CCC AUU UUG GCA CCA AAU ACG GCA GUA CAG Proteins that catalyze the formation or cleavage of nucleotide UAA UCG ACA AGU ACU GGA UGG CCA UAG UGG glycosidic linkages have been shown to exploit a number of CAC AAA AUA GGC CUC UAC CAG CGG UGU GGG mechanistically distinct reaction pathways (5–10). One extreme GUA ACC CAC AUC GCA GCA ACC). This ribozyme involves the stabilization of oxocarbenium-ion intermediates. performs the 4-thiouridine synthesis reaction with an apparent Hydrolytic enzymes, such as uracil DNA glycosylase and ricin 4S Ϫ Ϫ k ͞K Ura of 50 M 1⅐min 1, which is at least 108 times more A-chain, have been shown through experimental and quantum cat m efficient than the uncatalyzed reaction. 32P- and 33P-labeled mechanical studies to form an oxocarbenium ion that ultimately ribozyme was produced by T7 transcription with a final specific is quenched by water (9, 11, 12). In contrast, enzymes such as activity Ϸ9,600 GBq͞mmol (isotopes from New England Nu- pertussis toxin and many glycosyl transfer enzymes have been clear). Gel-purified RNA transcripts were attached to PRPP by shown to promote concerted reactions having low but apprecia- using preadenylated PRPP (AppRpp) and T4 RNA ligase [1–5 ble amounts of bond order during the chemical step (9). ␮M RNA, 40–50 ␮M isotopically labeled AppRpp, 50 mM The ability of RNA, with its four bulky functional groups, to ␮ ͞ Hepes (pH 8.3), 5 mM MgCl2, 3.3 mM DTT, 10 g ml BSA in perform glycosidic chemistry might intrinsically be more limited 8.3% glycerol, and 0.5 units͞␮l enzyme from Pharmacia in a 4-h than that of evolved protein catalysts. Thus an RNA catalyst might not be able to efficiently support the range of mechanisms used by protein enzymes, which possess a diverse set of functional groups This paper was submitted directly (Track II) to the PNAS office. and have been optimized through billions of years of evolution. A Abbreviations: PRPP, 5-phosphoribosyl 1-pyrophosphate; AppRpp, 5-adenylyl-PRPP; KIE, ki- fundamental difficulty faced by all catalytic systems promoting netic isotope effect; APM, [(N-acryloylamino)phenyl]mercuric chloride; 4SUra, 4-thiouracil. nucleotide synthesis is that the uncatalyzed rate of PRPP hydrolysis ‡To whom correspondence may be addressed. E-mail: [email protected] or dbartel@ greatly exceeds that of nucleotide synthesis, which occurs at an wi.mit.edu. undetectably low rate (1). Because hydrolysis of PRPP is likely to © 2003 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.2433147100 PNAS ͉ December 23, 2003 ͉ vol. 100 ͉ no. 26 ͉ 15393–15397 performed to generate the AppRpp used to synthesize the ribozyme-[1-1H]PRPP͞ribozyme-[1-12C]PRPP species. Analysis of Isotopic Purity. A 2.6-nmol sample of each isotopically labeled AppRpp synthesis was mixed with GpC and ApApC oligoribonucleotide mass standards in a matrix of 22.5 g͞liter 3-hydroxypicolinic acid, 2.5 g͞liter L-tartaric acid in 50% ace- tonitrile for matrix-assisted laser desorption͞ionization–time-of- flight (MALDI-TOF) analysis (Voyager instrument, PerSeptive Biosystems, Framingham, MA). Calibration was performed by using the oligoribonucleotide mass standards, which bracket the mass of AppRpp. As expected, the 13C- and 2H-labeled AppRpp samples were 1 atomic mass unit heavier than the unenriched AppRpp (predicted m͞z for lightest isotopic peak was 718.00 atomic mass units, observed was 718.08 atomic mass units; predicted m͞z for lightest isotopic peak of 13C- and 2H- substituted molecules was 719.00 atomic mass units, observed were 719.00 and 719.19 atomic mass units, respectively). To evaluate purity, MALDI-TOF spectra for each isotopic deriva- tive of AppRpp were first baseline subtracted and normalized, then aligned along the m͞z axis until the highest channel from each derivative was in register with underivatized AppRpp. The peak shapes were virtually identical and showed Ͻ5% variability from sample to sample in the regions diagnostic of potential Fig. 1. Two mechanistic extremes for ribozyme-promoted 4-thiouridine isotopic contamination. From this analysis we infer that the synthesis. (a) A stepwise dissociative mechanism (International Union of Pure isotopic purity of each AppRpp synthesis was Ͼ95%. and Applied Chemistry nomenclature: DNϩAN or DN*AN) involving the dis- placement of pyrophosphate (PPi), and the formation of an oxocarbenium-ion Kinetic Analysis. RNA pairs were heated to 80°C in water and intermediate, followed by nucleophilic addition of 4SUra and the production cooled to room temperature before addition of buffer. Ri- 4S of 4-thiouridine. (b) A concerted reaction (ANDN) involving the attack of Ura bozyme reactions were performed with 1 mM 4SUra in 50 mM and concerted displacement of pyrophosphate. ⅐ ͞ ͞ Tris HCl (pH 7.6) 25 mM MgCl2 150 mM KCl at 23°C. Reacted ribozyme was purified away from unreacted ribozyme by using ͞ incubation at room temperature]. Ligation was terminated by the [(N-acryloylamino)phenyl]mercuric chloride (APM) polyacryl- addition of EDTA followed by phenol͞chloroform extraction, amide gels (1). To achieve approximately equal yield of reacted ethanol precipitation, and resuspension in water. To ensure ribozyme product for each time point, smaller aliquots were equal treatment of both species of a dual-labeled pair, the loaded for the later time points. Scintillation counting was then 32 33 appropriate dual-labeled species were mixed together to form used to determine R(t), the relative ratios of Pto Pinthe pairs no.