The Tetrahymena Ribozyme Cleaves a 5‘-Methylene Phosphonate Monoester ∼10²-fold Faster Than a Normal Phosphate Diester:  Implications for Enzyme Catalysis of Phosphoryl Transfer Reactions

Abstract

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by <5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate mono...

Novel isosteric, isopolar phosphonate analogs of oligonucleotides: Preparation and properties

“…Oligomers 16-18 and 4 0 0 -C-methyl phosphodiester dT 15 Oligomer 19 The solid-phase syntheses of phosphonate oligomers 16-18 were performed from the 5 0 -end on a GenSyn (IOCB Prague) synthesizer using CPG support modified with 2 0 -deoxy-3 0 -O-DMTr-thymidine-5 0 -hemisuccinate (0.5 mol scale). The preparation of oligonucleotide 16 with all-phosphonate backbone was carried out with the monomer 15a (and its homooligomeric counterpart with ''phosphonate-down'' -D-erythro configuration likewise with 15b; not shown) by the phosphotriester method 13,27,28 whereas the oligomers with mixed phosphonate-phosphodiester linkages 17 and 18 ( Figure 2) were prepared by alternating incorporation of a ''reversed'' amidite, the 3 0 -O-dimethoxytritylthymidine 5 0 -O-(cyanoethyl-diisopropylphosphoramidite), and the phosphonate monomer 15a or 15b using the phosphoramidite and phosphotriester protocols in the respective elongation steps.…”

“…Mixing curves (not shown) of oligomers 17 and 19 with dA 15 (modified dT 15 :dA 15 ) exhibited flat minima, indicating the possible presence of both duplex and triplex structures. Because in this case the method did not allow for unambiguous determination of either simple stoichiometry of complex(es) or an equilibrium concentration of the duplexes and triplexes, the molecularity of the complexes could not be determined.…”

“…14 The C3 0 -O-P-CH 2 -CH 2 -C4 00 linkage of these oligomers was found to be resistant toward calf spleen phosphodiesterase but unstable toward snake venom exonuclease. 13 These phosphonate oligomers did not found a broader biological exploitation but, recently, Liao et al 15 reported on a faster cleavage of a 5 0 -methylene phosphonate unit-containing oligomer by Tetrahymena ribozyme in comparison with unmodified substrate. Another paper showed that the analogue of dTTP and ADP containing likewise a methylene group in place of the 5 0 oxygen atom were substrates for reverse transcriptase 16 and PNPase, 17 respectively.…”

A kinetic and thermodynamic framework for the Azoarcus group I ribozyme reaction

“…A deleterious impact on G binding to the Tetrahymena ribozyme is observed for a "dangling" 3 ′ U residue of S when the P1 helix is docked in active site (Liao et al 2001;Karbstein et al 2007), and this effect may be analogous to the observed inhibitory effect of the unpaired IGS extension or resulting unpaired 3 ′ -tail of CAUA 5 on the S-bound L-6A Azoarcus ribozyme. Fortuitous interactions may be common when residues are positioned within RNA "pockets," especially given the relatively simple hydrogen bonding and stacking recognition patterns in RNA (e.g., Peracchi et al 1996;Karbstein et al 2007).…”

Understanding the transition states of phosphodiester bond cleavage: Insights from heavy atom isotope effects

“…34 Comparison of the rate of methylene monoesters shows that changing this oxygen to carbon has only a small effect on reaction rate. A reexamination of previous physical organic analyses indicates that this group undergoes little charge buildup in the transition state; thus, enzyme interactions with this group are not expected to provide significant catalytic advantage.…”