Allosterically activated Diels-Alder catalysis by a ribozyme

We describe the allosteric control of Diels-Alder reactions by a small organic effector, theophylline. This is achieved by converting a Diels-Alder ribozyme into an allosterically regulated system. In contrast to other published systems, we have a bond-forming reaction with two small-molecule substrates and multiple turnover. This system could be very attractive for the development of assays for a variety of analytes and can be regarded as a prototype of fully synthetic signaling cascades.     

Nucleobase participation in ribozyme catalysis

We constructed a modified form of the VS ribozyme containing an imidazole ring in place of adenine at position 756. The novel ribozyme is active in both cleavage and ligation reactions. The reaction is efficient, although relatively slow. The results are consistent with a role for nucleobase catalysis in the catalytic mechanism of this ribozyme.     

Solvent structure and hammerhead ribozyme catalysis

Although the hammerhead ribozyme is regarded as a prototype for understanding RNA catalysis, the mechanistic roles of associated metal ions and water molecules in the cleavage reaction remain controversial. We have investigated the catalytic potential of observed divalent metal ions and water molecules bound to a 2 A structure of the full-length hammerhead ribozyme by using X-ray crystallography in combination with molecular dynamics simulations. A single Mn(2+) is observed to bind directly to the A9 phosphate in the active site, accompanying a hydrogen-bond network involving a well-ordered water molecule spanning N1 of G12 (the general base) and 2'-O of G8 (previously implicated in general acid catalysis) that we propose, based on molecular dynamics calculations, facilitates proton transfer in the cleavage reaction. Phosphate-bridging metal interactions and other mechanistic hypotheses are also tested with this approach.     

Probing general base catalysis in the hammerhead ribozyme

Recent structural and computational studies have shed new light on the catalytic mechanism and active site structure of the RNA cleaving hammerhead ribozyme. Consequently, specific ribozyme functional groups have been hypothesized to be directly involved in general/acid base catalysis. In order to test this hypothesis, we have developed an affinity label to identify the functional general base in the S. mansoni hammerhead ribozyme. The ribozyme was reacted with a substrate analogue bearing a 2'-bromoacetamide group in place of the nucleophilic 2'-hydroxyl group which would normally be deprotonated by a general base. The electrophilic 2'-bromoacetamide group is poised to alkylate the general base, which is subsequently identified by footprinting analysis. Herein, we demonstrate alkylation of N1 of G12 in the hammerhead ribozyme in a pH and [Mg(2+)] dependent manner that is consistent with the native cleavage reaction. These results provide substantial evidence that deprotonated N1 of G12 functions directly as a general base in the hammerhead ribozyme; moreover, our experiments provide evidence that the pKa of G12 is perturbed downward in the context of the active site structure. We also observed other pH-independent alkylations, which do not appear to reflect the catalytic mechanism, but offer further insight into ribozyme conformation and structure.     

Diels-Alder ribozyme catalysis: a computational approach

A computational comparison of the Diels-Alder reaction of a maleimide and an anthracene in water and the active site of the ribozyme Diels-Alderase is reported. During the course of the catalyzed reaction, the maleimide is held in the hydrophobic pocket while the anthracene approaches to the maleimide through the back passage of the active site. The active site is so narrow that the anthracene has to adopt a tilted approach angle toward maleimide. The conformation of the active site changes marginally at different states of the reaction. Active site dynamics contribution to catalysis has been ruled out. The active site stabilizes the product more than the transition state (TS). The reaction coordinates of the ribozyme reaction in TS, RC1-CD1 and RC4-CD2, are 2.35 and 2.33 A, respectively, compared to 2.37 and 2.36 A in water. The approach angle of anthracene toward maleimide is twisted by 18 degrees in the TS structure of ribozyme reaction while no twisted angle is found in TS of the reaction in water. The free energy barriers for reactions in both ribozyme and water were obtained by umbrella sampling combined with SCCDFTB/MM. The calculated free energy barriers for the ribozyme and water reactions are in good agreement with the experimental values. As expected, Mulliken charges of the atoms involved in the ribozyme reaction change in a similar manner as that of the reaction in water. The proficiency of the Diels-Alder ribozyme reaction originates from the active site holding the two reactants in reactive conformations, in which the reacting atoms are brought together in van der Waals distances and reactants approach to each other at an appropriate angle.     

A hydrogen-bonding triad stabilizes the chemical transition state of a group I ribozyme

BACKGROUND: The group I intron is an RNA enzyme capable of efficiently catalyzing phosphoryl-transfer reactions. Functional groups that stabilize the chemical transition state of the cleavage reaction have been identified, but they are all located within either the 5'-exon (P1) helix or the guanosine cofactor, which are the substrates of the reaction. Functional groups within the ribozyme active site are also expected to assist in transition-state stabilization, and their role must be explored to understand the chemical basis of group I intron catalysis. RESULTS: Using nucleotide analog interference mapping and site-specific functional group substitution experiments, we demonstrate that the 2'-OH at A207, a highly conserved nucleotide in the ribozyme active site, specifically stabilizes the chemical transition state by approximately 2 kcal mol-1. The A207 2'-OH only makes its contribution when the U(-1) 2'-OH immediately adjacent to the scissile phosphate is present, suggesting that the 2'-OHs of A207 and U(-1) interact during the chemical step. CONCLUSIONS: These data support a model in which the 3'-oxyanion leaving group of the transesterification reaction is stabilized by a hydrogen-bonding triad consisting of the 2'-OH groups of U(-1) and A207 and the exocyclic amine of G22. Because all three nucleotides occur within highly conserved non-canonical base pairings, this stabilization mechanism is likely to occur throughout group I introns. Although this mechanism utilizes functional groups distinctive of RNA enzymes, it is analogous to the transition states of some protein enzymes that perform similar phosphoryl-transfer reactions.     

A role for a single-stranded junction in RNA binding and specificity by the Tetrahymena group I ribozyme

We have investigated the role of a single-stranded RNA junction, J1/2, that connects the substrate-containing P1 duplex to the remainder of the Tetrahymena group I ribozyme. Single-turnover kinetics, fluorescence anisotropy, and single-molecule fluorescence resonance energy transfer studies of a series of J1/2 mutants were used to probe the sequence dependence of the catalytic activity, the P1 dynamics, and the thermodynamics of docking of the P1 duplex into the ribozyme's catalytic core. We found that A29, the center A of three adenosine residues in J1/2, contributes 2 orders of magnitude to the overall ribozyme activity, and double-mutant cycles suggested that J1/2 stabilizes the docked state of P1 over the undocked state via a tertiary interaction involving A29 and the first base pair in helix P2 of the ribozyme, A31·U56. Comparative sequence analysis of this group I intron subclass suggests that the A29 interaction sets one end of a molecular ruler whose other end specifies the 5'-splice site and that this molecular ruler is conserved among a subclass of group I introns related to the Tetrahymena intron. Our results reveal substantial functional effects from a seemingly simple single-stranded RNA junction and suggest that junction sequences may evolve rapidly to provide important interactions in functional RNAs.     

A phosphoramidate substrate analog is a competitive inhibitor of the Tetrahymena group I ribozyme

BACKGROUND: Phosphoramidate oligonucleotide analogs containing N3'-P5' linkages share many structural properties with natural nucleic acids and can be recognized by some RNA-binding proteins. Therefore, if the N-P bond is resistant to nucleolytic cleavage, these analogs may be effective substrate analog inhibitors of certain enzymes that hydrolyze RNA. We have explored the ability of the Tetrahymena group I intron ribozyme to bind and cleave DNA and RNA phosphoramidate analogs. RESULTS: The Tetrahymena group I ribozyme efficiently binds to phosphoramidate oligonucleotides but is unable to cleave the N3'-P5' bond. Although it adopts an A-form helical structure, the deoxyribo-phosphoramidate analog, like DNA, does not dock efficiently into the ribozyme catalytic core. In contrast, the ribo-phosphoramidate analog docks similarly to the native RNA substrate, and behaves as a competitive inhibitor of the group I intron 5' splicing reaction. CONCLUSIONS: Ribo-N3'-P5' phosphoramidate oligonucleotides are useful tools for structural and functional studies of ribozymes as well as protein-RNA interactions.     

The role of the cleavage site 2'-hydroxyl in the Tetrahymena group I ribozyme reaction

BACKGROUND: The 2'-hydroxyl of U preceding the cleavage site, U(-1), in the Tetrahymena ribozyme reaction contributes 10(3)-fold to catalysis relative to a 2'-hydrogen atom. Previously proposed models for the catalytic role of this 2'-OH involve coordination of a catalytic metal ion and hydrogen-bond donation to the 3'-bridging oxygen. An additional model, hydrogen-bond donation by the 2'-OH to a nonbridging reactive phosphoryl oxygen, is also consistent with previous results. We have tested these models using atomic-level substrate modifications and kinetic and thermodynamic analyses. RESULTS: Replacing the 2'-OH with -NH(3)(+) increases the reaction rate approximately 60-fold, despite the absence of lone-pair electrons on the 2'-NH(3)(+) group to coordinate a metal ion. Binding and reaction of a modified oligonucleotide substrate with 2'-NH(2) at U(-1) are unaffected by soft-metal ions. These results suggest that the 2'-OH of U(-1) does not interact with a metal ion. The contribution of the 2'-moiety of U(-1) is unperturbed by thio substitution at either of the nonbridging oxygens of the reactive phosphoryl group, providing no indication of a hydrogen bond between the 2'-OH and the nonbridging phosphoryl oxygens. In contrast, the 10(3)-fold catalytic advantage of 2'-OH relative to 2'-H is eliminated when the 3'-bridging oxygen is replaced by sulfur. As sulfur is a weaker hydrogen-bond acceptor than oxygen, this effect suggests a hydrogen-bonding interaction between the 2'-OH and the 3'-bridging oxygen. CONCLUSIONS: These results provide the first experimental support for the model in which the 2'-OH of U(-1) donates a hydrogen bond to the neighboring 3'-bridging oxygen, thereby stabilizing the developing negative charge on the 3'-oxygen in the transition state.     

Essential role of an active-site guanine in glmS ribozyme catalysis

The glmS ribozyme is a catalytic riboswitch that is activated for endonucleolytic cleavage by the coenzyme glucosamine-6-phosphate. Using kinetic assays and X-ray crystallography, we identify an active-site mutation of a conserved guanine that abolishes catalysis without perturbing coenzyme binding. Our results provide evidence that coenzyme function requires a specific nucleobase to interact with the nucleophile of the cleavage reaction.     

An unusual pH-independent and metal-ion-independent mechanism for hairpin ribozyme catalysis

Background: Hairpin ribozymes (RNA enzymes) catalyze the same chemical reaction as ribonuclease A and yet RNAs do not usually have functional groups analogous to the catalytically essential histidine and lysine sidechains of protein ribonucleases. Some RNA enzymes appear to recruit metal ions to act as Lewis acids in charge stabilization and metal-bound hydroxide for general base catalysis, but it has been reported that the hairpin ribozyme functions in the presence of metal ion chelators. This led us to investigate whether the hairpin ribozyme exploits a metal-ion-independent catalytic strategy.Results: Substitution of sulfur for nonbridging oxygens of the reactive phosphate of the hairpin ribozyme has small, stereospecific and metal-ionindependent effects on cleavage and ligation mediated by this ribozyme. Cobalt hexammine, an exchange-inert metal complex, supports full hairpin ribozyme activity, and the ribozyme's catalytic rate constants display only a shallow dependence on pH.Conclusions: Direct metal ion coordination to phosphate oxygens is not essential for hairpin ribozyme catalysis and metal-bound hydroxide does not serve as the general base in this catalysis. Several models might account for the unusual pH and metal ion independence: hairpin cleavage and ligation might be limited by a slow conformational change; a pH-independent or metalcation-independent chemical step, such as breaking the 5′ oxygen-phosphorus bond, might be rate determining; or finally, functional groups within the ribozyme might participate directly in catalytic chemistry. Whichever the case, the hairpin ribozyme appears to employ a unique strategy for RNA catalysis.     

Separate metal requirements for loop interactions and catalysis in the extended hammerhead ribozyme

An extended hammerhead ribozyme derived from Schistosoma mansoni, including conserved loops in stems I and II, has been examined to directly monitor the relationship between docking of loops and its activity using site-directed spin labeling (SDSL) and EPR spectroscopy. Dynamics with EPR spectroscopy and fast-quench kinetics measurements have shown that the docking of stems I and II occurs at low Mg2+ concentrations ([Mg2+]1/2,dock = 0.7 mM, 0.1 M NaCl), but a much weaker Mg2+ interaction ([Mg2+]1/2,cat approximately 90 mM) increases activity to very high maximum rates of approximately 1 s-1 at 0.1 M Na+ and pH 7.0.     

Role of Mg2+ in hammerhead ribozyme catalysis from molecular simulation

Molecular dynamics simulations have been performed to investigate the role of Mg2+ in the full-length hammerhead ribozyme cleavage reaction. In particular, the aim of this work is to characterize the binding mode and conformational events that give rise to catalytically active conformations and stabilization of the transition state. Toward this end, a series of eight 12 ns molecular dynamics simulations have been performed with different divalent metal binding occupations for the reactant, early and late transition state using recently developed force field parameters for metal ions and reactive intermediates in RNA catalysis. In addition, hybrid QM/MM calculations of the early and late transition state were performed to study the proton-transfer step in general acid catalysis that is facilitated by the catalytic Mg2+ ion. The simulations suggest that Mg2+ is profoundly involved in the hammerhead ribozyme mechanism both at structural and catalytic levels. Binding of Mg2+ in the active site plays a key structural role in the stabilization of stem I and II and to facilitate formation of near attack conformations and interactions between the nucleophile and G12, the implicated general base catalyst. In the transition state, Mg2+ binds in a bridging position where it stabilizes the accumulated charge of the leaving group while interacting with the 2'OH of G8, the implicated general acid catalyst. The QM/MM simulations provide support that, in the late transition state, the 2'OH of G8 can transfer a proton to the leaving group while directly coordinating the bridging Mg2+ ion. The present study provides evidence for the role of Mg2+ in hammerhead ribozyme catalysis. The proposed simulation model reconciles the interpretation of available experimental structural and biochemical data, and provides a starting point for more detailed investigation of the chemical reaction path with combined QM/MM methods.     

Quantum mechanical/molecular mechanical simulation study of the mechanism of hairpin ribozyme catalysis

The molecular mechanism of hairpin ribozyme catalysis is studied with molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential with a recently developed semiempirical AM1/d-PhoT model for phosphoryl transfer reactions. Simulations are used to derive one- and two-dimensional potentials of mean force to examine specific reaction paths and assess the feasibility of proposed general acid and base mechanisms. Density-functional calculations of truncated active site models provide complementary insight to the simulation results. Key factors utilized by the hairpin ribozyme to enhance the rate of transphosphorylation are presented, and the roles of A38 and G8 as general acid and base catalysts are discussed. The computational results are consistent with available experimental data, provide support for a general acid/base mechanism played by functional groups on the nucleobases, and offer important insight into the ability of RNA to act as a catalyst without explicit participation by divalent metal ions.     

Tightening of active site interactions en route to the transition state revealed by single-atom substitution in the guanosine-binding site of the Tetrahymena group I ribozyme

Protein enzymes establish intricate networks of interactions to bind and position substrates and catalytic groups within active sites, enabling stabilization of the chemical transition state. Crystal structures of several RNA enzymes also suggest extensive interaction networks, despite RNA's structural limitations, but there is little information on the functional and the energetic properties of these inferred networks. We used double mutant cycles and presteady-state kinetic analyses to probe the putative interaction between the exocyclic amino group of the guanosine nucleophile and the N7 atom of residue G264 of the Tetrahymena group I ribozyme. As expected, the results supported the presence of this interaction, but remarkably, the energetic penalty for introducing a CH group at the 7-position of residue G264 accumulates as the reaction proceeds toward the chemical transition state to a total of 6.2 kcal/mol. Functional tests of neighboring interactions revealed that the presence of the CH group compromises multiple contacts within the interaction network that encompass the reactive elements, apparently forcing the nucleophile to bind and attack from an altered, suboptimal orientation. The energetic consequences of this indirect disruption of neighboring interactions as the reaction proceeds demonstrate that linkage between binding interactions and catalysis hinges critically on the precise structural integrity of a network of interacting groups.     

An active-site guanine participates in glmS ribozyme catalysis in its protonated state

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.     

Amide group catalysis of ester hydrolysis

Hydrolysis of phenyl-N-acetylanthranilate 1 (Ar = Ph) to N-acetyl anthranilie acid 3 is base catalysed and occurs through the intermediacy of 2-methyl-3,1-benzoxazin-4-one 4. Above pH 6 the formation of 4 from 1 is faster than the base (or acid) catalysed ring opening of 4 to 3. Electron-withdrawing substituents in the ester moiety (e.g. 1, Ar = p-NO₂C₆H₄) aid cyclization to 4 relative to direct hydroxide catalyzed hydrolysis (to 3). Concomitantly, neutral amide participation is observed so that cyclization of 1 (Ar = p-NO₂, m-NO₂, m-Cl, p-ClC₆H₄) occurs even in acidic solution; the Hammett ? values for neutral and base catalysed cyclizations are compared. Hydrolysis of methyl-N-acetylanthranilate 6 to 3 occurs slowly in base, possibly without the formation of the intermediate 4.     

Pseudorotation barriers of biological oxyphosphoranes: a challenge for simulations of ribozyme catalysis

Pseudorotation reactions of biologically relevant oxyphosphoranes were studied by using density functional and continuum solvation methods. A series of 16 pseudorotation reactions involving acyclic and cyclic oxyphosphoranes in neutral and monoanionic (singly deprotonated) forms were studied, in addition to pseudorotation of PF5. The effect of solvent was treated by using three different solvation models for comparison. The barriers to pseudorotation ranged from 1.5 to 8.1 kcal mol(-1) and were influenced systematically by charge state, apicophilicity of ligands, intramolecular hydrogen bonding, cyclic structure and solvation. Barriers to pseudorotation for monoanionic phosphoranes occur with the anionic oxo ligand as the pivotal atom, and are generally lower than for neutral phosphoranes. The OCH3 groups were observed to be more apicophilic than OH groups, and hence pseudorotations that involve axial OCH3/equatorial OH exchange had higher reaction and activation free energy values. Solvent generally lowered barriers relative to the gas-phase reactions. These results, together with isotope 18O exchange experiments, support the assertion that dianionic phosphoranes are not sufficiently long-lived to undergo pseudorotation. Comparison of the density functional results with those from several semiempirical quantum models highlight a challenge for new-generation hybrid quantum mechanical/molecular mechanical potentials for non-enzymatic and enzymatic phosphoryl transfer reactions: the reliable modeling of pseudorotation processes.