Interactions In Transition State Research Articles

Transition-State Interactions Revealed in Purine Nucleoside Phosphorylase by Binding Isotope Effects

The binding of [5‘-3H]inosine to human purine nucleoside phosphorylase results in an equilibrium binding isotope effect (BIE) of 1.5%, and transition state formation causes an intrinsic KIE of 4.7%. These values reflect atomic vibrational distortions in the 5‘-C−H bonds upon formation of the Michaelis complex and transition state. The degree of atomic distortion for catalysis is compared to that for binding of transition state analogues. Similar radiolabeled forms of the transition-state analogues ImmH and DADMe-ImmH gave large 5‘-3H BIEs of 12.6% and 29.2%, respectively. Greater bond distortions occur upon complex formation with transition-state analogues, supporting weaker distortional forces at the transition state than in the formation of complexes with transition-state analogues.

Kinetic isotope effects for alkaline phosphatase reactions: implications for the role of active-site metal ions in catalysis.

Enzyme-catalyzed phosphoryl transfer reactions have frequently been suggested to proceed through transition states that are altered from their solution counterparts, with the alterations presumably arising from interactions with active-site functional groups. In particular, the phosphate monoester hydrolysis reaction catalyzed by Escherichia coli alkaline phosphatase (AP) has been the subject of intensive scrutiny. Recent linear free energy relationship (LFER) studies suggest that AP catalyzes phosphate monoester hydrolysis through a loose transition state, similar to that in solution. To gain further insight into the nature of the transition state and active-site interactions, we have determined kinetic isotope effects (KIEs) for AP-catalyzed hydrolysis reactions with several phosphate monoester substrates. The LFER and KIE data together provide a consistent picture for the nature of the transition state for AP-catalyzed phosphate monoester hydrolysis and support previous models suggesting that the enzymatic transition state is similar to that in solution. Moreover, the KIE data provides unique information regarding specific interactions between the transition state and the active-site Zn2+ ions. These results provide strong support for a model in which electrostatic interactions between the bimetallo Zn2+ site and a nonbridging phosphate ester oxygen atom make a significant contribution to the large rate enhancement observed for AP-catalyzed phosphate monoester hydrolysis.

Transition‐State Energy and Position along the Reaction Coordinate in an Extended Activation Strain Model

We investigate palladium-induced activation of the C-H, C-C, C-F, and C-Cl bonds in methane, ethane, cyclopropane, fluoromethane, and chloromethane, using relativistic density functional theory (DFT) at ZORA-BLYP/TZ2P. Our purpose is to arrive at a qualitative understanding, based on accurate calculations, of the trends in activation barriers and transition state (TS) geometries (e.g. early or late along the reaction coordinate) in terms of the reactants' properties. To this end, we extend the activation strain model (in which the activation energy Delta E(not equal) is decomposed into the activation strain Delta E(not equal)(strain) of the reactants and the stabilizing TS interaction Delta E(not equal)(int) between the reactants) from a single-point analysis of the TS to an analysis along the reaction coordinate zeta, that is, Delta E(zeta)=Delta E(strain)(zeta)+Delta E(int)(zeta). This extension enables us to understand qualitatively, trends in the position of the TS along zeta and, therefore, the values of the activation strain Delta E(not equal)(strain)=Delta E(strain)(zeta(TS)) and TS interaction Delta E(not equal)(int)=Delta E(int)(zeta(TS)) and trends therein. An interesting insight that emerges is that the much higher barrier of metal-mediated C-C versus C-H activation originates from steric shielding of the C-C bond in ethane by C-H bonds. Thus, before a favorable stabilizing interaction with the C-C bond can occur, the C-H bonds must be bent away, which causes the metal-substrate interaction Delta E(int)(zeta) in C-C activation to lag behind. Such steric shielding is not present in the metal-mediated activation of the C-H bond, which is always accessible from the hydrogen side. Other phenomena that are addressed are anion assistance, competition between direct oxidative insertion (OxIn) versus the alternative S(N)2 pathway, and the effect of ring strain.

Kinetics and mechanism of unimolecular heterolysis of cage-like compounds: XIX. Effect of the nucleofuge nature on the activation parameters of heterolysis of 1-halo-1-methylcyclohexanes in cyclohexane. Heterolysis rate ratio in aprotic and protic solvents

Heterolysis of 1-bromo-1-methylcyclohexane in cyclohexane (E1 reaction) involves solvation of the transition state (ΔS≡ = −81 J mol−1K−1), while heterolysis of 1-chloro-1-methylcyclohexane is characterized by desolvation of the transition state (ΔS≡ = 92 J mol−1K−1). The probability for the formation of transition state (interaction between cationoid intermediate and solvent cavity) increases in the first case due to enhanced stability of the solvated intermediate, and in the second, due to reduction in its size. The bromide/chloride heterolysis rate ratio decreases as the ionizing power of aprotic solvent decreases and that of protic solvent increases.

Correlation between Protein Stability Cores and Protein Folding Kinetics: A Case Study on Pseudomonas aeruginosa Apo-Azurin

This paper reports a combined computational and experimental study of the correlation between protein stability cores and folding kinetics. An empirical potential function was developed, and it was used for analyzing interaction energies among secondary structure elements. Studies on a beta sandwich protein, Pseudomonas aeruginosa azurin, showed that the computationally identified substructure with the strongest interactions in the native state is identical to the "interlocked pair" of beta strands, an invariant motif found in most sandwich-like proteins. Moreover, previous and new in vitro folding results revealed that the identified substructure harbors most residues that form native-like interactions in the folding transition state. These observations demonstrate that the potential function is effective in revealing the relative strength of interactions among various protein parts; they also strengthen the suggestion that the most stable regions in native proteins favor stable interactions early during folding.

The Properties of Steric Gate Mutants Reveal Different Constraints within the Active Sites of Y-family and A-family DNA Polymerases

Y-family (lesion-bypass) DNA polymerases show the same overall structural features seen in other members of the polymerase superfamily, yet their active sites are more open, with fewer contacts to the DNA and nucleotide substrates. This raises the question of whether analogous active-site side chains play equivalent roles in the bypass polymerases and their classical DNA polymerase counterparts. In Klenow fragment, an A-family DNA polymerase, the steric gate side chain (Glu710) not only prevents ribonucleotide incorporation but also plays an important role in discrimination against purine-pyrimidine mispairs. In this work we show that the steric gate (Phe12) of the Y-family polymerase Dbh plays a very minor role in fidelity, despite its analogous role in sugar selection. Using ribonucleotide discrimination to report on the positioning of a mispaired dNTP, we found that the pyrimidine of a Pu-dPyTP nascent mispair occupies a similar position to that of a correctly paired dNTP in the Dbh active site, whereas in Klenow fragment the mispaired dNTP sits higher in the active site pocket. If purine-pyrimidine mispairs adopt the expected wobble geometry, the difference between the two polymerases can be attributed to the binding of the templating base, with the looser binding site of Dbh permitting a variety of template conformations with only minimal adjustment at the incoming dNTP. In Klenow fragment the templating base is more rigidly held, so that changes in base pair geometry would affect the dNTP position, allowing the Glu710 side chain to serve as a sensor of nascent mispairs.

Quantitative Classification of Covalent and Noncovalent H-Bonds

The covalent nature of interactions within various hydrogen bonded molecular aggregates has been characterized by the two entirely different computational methods: Bader analysis of the electron density and variation-perturbation partitioning of the intermolecular interaction energy. Analysis of 34 complexes representing different types of hydrogen bonds indicates that the proton-acceptor distance approximately 1.8 A and the ratio of delocalization and electrostatic terms approximately 0.45 constitutes approximately a borderline between covalent and noncovalent hydrogen bonds. The latter ratio could be used to characterize quantitatively the degree of the covalent nature of transition state interactions with active site residues, a quantity essential for an enzyme catalytic activity.

Functional Identification of Catalytic Metal Ion Binding Sites within RNA

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-S P phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.

Interpretation of Protein Folding ψ Values

The structural characterization of transition states is essential for understanding the mechanism of protein folding. Analyzing the effect of mutations on protein stability and folding kinetics in phi-value analysis is commonly used to gain information about the presence of side-chain interactions in transition states. Recently, specific binding of ligands to engineered binding sites was applied to monitor the formation of local structures in transition states (psi analysis). A surprising result from psi analysis was the presence of parallel folding pathways in all reported studies and a major discrepancy between phi and psi values measured in the same protein. Here, we show that psi values cannot be analyzed in the same way as other rate-equilibrium free energy relationships due to the involvement of bimolecular reactions that may have different dissociation constants for the native, unfolded and transition state. As a consequence, psi values reflect the relative binding energy (kappa) of the transition state only for the extreme values of kappa=0 or kappa=1. In all other cases, non-linear rate-equilibrium free-energy relationships (Leffler plots) are observed. This apparently indicates the presence of parallel folding pathways even if folding occurs over a single homogeneous transition state. Consequently, the results from Leffler plots do not yield information about the structural properties of the transition state. This explains the lack of agreement between results from psi analysis and other methods used to characterize protein folding transition states. We further show that the same considerations apply for the analysis of the effect of pH on protein folding.

Activation of C–H, C–C and C–I bonds by Pd and cis-Pd(CO) 2I 2. Catalyst–substrate adaptation

We have studied the oxidative addition reactions of methane and ethane C–H, ethane C–C and iodomethane C–I bonds to Pd and cis-Pd(CO) 2I 2 at the ZORA-BP86/TZ(2)P level of relativistic density functional theory (DFT). Our purpose, besides exploring these particular model reactions, is to understand how the mechanism of bond activation changes as the catalytically active species changes from a simple, uncoordinated metal atom to a metal–ligand coordination complex. For both Pd and cis-Pd(CO) 2I 2, direct oxidative insertion (OxIn) is the lowest-barrier pathway whereas nucleophilic substitution (S N2) is highly endothermic, and therefore not competitive. Introducing the ligands, i.e., going from Pd to cis-Pd(CO) 2I 2, causes a significant increase of the activation and reaction enthalpies for oxidative insertion and takes away the intrinsic preference of Pd for C–I over C–H activation. Obviously, cis-Pd(CO) 2I 2 is a poor catalyst in terms of activity as well as selectivity for one of the three bonds studied. However, its exploration sheds light on features in the process of catalytic bond activation associated with the increased structural and mechanistic complexity that arises if one goes from a monoatomic model catalysts to a more realistic transition-metal complex. First, in the transition state (TS) for oxidative insertion, the C–X bond to be activated can have, in principle, various different orientations with respect to the square-planar cis-Pd(CO) 2I 2 complex, e.g., C–X or X–C along an I–Pd–CO axis, or in between two I–Pd–CO axes. Second, at variance to the uncoordinated metal atom, the metal complex may be deformed due to the interaction with the substrate. This leads to a process of mutual adjustment of catalyst and substrate that we designate catalyst–substrate adaptation. The latter can be monitored by the Activation Strain model in which activation energies Δ E ≠ are decomposed into the activation strain Δ E strain ≠ of and the stabilizing TS interaction Δ E int ≠ between the reactants in the activated complex: Δ E ≠ = Δ E strain ≠ + Δ E int ≠ .

Activation of H-H, C-H, C-C and C-Cl Bonds by Pd and PdCl(-). Understanding Anion Assistance in C-X Bond Activation.

To understand the mechanism of anion assistance in palladium-catalyzed H-H, C-H, C-C and C-Cl bond activation, several mechanistic pathways for oxidative addition of Pd and PdCl(-) to H2 (H-H), CH4 (C-H), C2H6 (C-C and C-H) and CH3Cl (C-Cl) were studied uniformly at the ZORA-BP86/TZ(2)P level of relativistic nonlocal density functional theory (DFT). Oxidative addition of the neutral, uncoordinated Pd atom proceeds, as reported earlier, via direct oxidative insertion (ΔH(⧧)298 is -22 to 10 kcal/mol), whereas straight SN2 substitution (yielding, e.g., PdCH3(+) + X(-)) is highly endothermic (144-237 kcal/mol) and thus not competitive. Anion assistance (i.e., going from Pd to PdCl(-)) lowers all activation barriers and increases the exothermicity of all model reactions studied. The effect is however selective: it favors the highly endothermic SN2 mechanism over direct oxidative insertion (OxIn). Activation enthalpies ΔH(⧧)298 for oxidative insertion of PdCl(-) increase along C-H (-14.0 and -13.5 kcal/mol for CH4 and C2H6) ≈ C-Cl (-11.2 kcal/mol) < C-C (6.4 kcal/mol), i.e., essentially in the same order as for neutral Pd. Interestingly, in case of PdCl(-) + CH3Cl, the two-step mechanism of SN2 substitution followed by leaving-group rearrangement becomes the preferred mechanism for oxidative addition. The highest overall barrier of this pathway (-20.2 kcal/mol) drops below the barrier for direct oxidative insertion (-11.2 kcal/mol). The effect of anion assistance is analyzed using the Activation Strain model in which activation energies ΔE(⧧) are decomposed into the activation strain ΔE(⧧)strain of and the stabilizing transition state (TS) interaction ΔE(⧧)int between the reactants in the activated complex: ΔE(⧧) = ΔE(⧧)strain + ΔE(⧧)int. For each type of activated bond and reaction mechanism, the activation strain ΔE(⧧)strain adopts characteristic values which differ only moderately, within a relatively narrow range, between corresponding reactions of Pd and PdCl(-). The lowering of activation barriers through anion assistance is caused by the TS interaction ΔE(⧧)int becoming more stabilizing.

The influence of constitutional isomerism and change on molecular recognition processes.

Three constitutionally isomeric bis(naphthylmethyl)ammonium ions, in which the two naphthyl groups are substituted 1) both at their 1-positions, 2) one at its 1-position and the other at its 2-position, and 3) both at their 2-positions, have been investigated separately in solution for their propensities to undergo spontaneous self-assembly with three different [24]crown-8 derivatives, namely, pyrido[24]crown-8 (P24C8), dipyrido[24]crown-8 (DP24C8) and dibenzo[24]crown-8 (DB24C8), in turn to form [2]pseudorotaxanes. The strengths of the 1:1 complexes depend on the composition of the secondary dialkylammonium ions and on the nature of the crown ether hosts; generally, as far as the guest cation is concerned, the 1/1- and 2/2-isomers form stronger complexes, as indicated by stability constant measurements, than the 1/2-isomer and, as far as the crown ethers are concerned, the more flexible P24C8 is a much more efficient host than either DP24C8 or DB24C8. The rates of formation of the [2]pseudorotaxanes are fast (i.e., taking no more than a few minutes) in solution with the exception of one case, that is, in which the crown ether host is DB24C8 and the guest cation is the 1/1-isomer, when it can take upwards of one month for the complexation-decomplexation equilibrium to be established at room temperature. In all cases, the equilibrium between complexed and uncomplexed species is slow on the NMR timescale, allowing the determination of stability constants to be made readily using the single-point method. X-ray crystallography and molecular modeling have been used to gain insight into ground and transition state interactions, respectively, in some of the [2]pseudorotaxanes. The relative stabilities of the three [2]pseudorotaxanes formed by each guest cation in the presence of the three crown ether hosts were also evaluated in solution by competition experiments that were monitored by (1)H NMR spectroscopy. By and large the results of the competition experiments could be predicted on the basis of the derived stability constants for the individual [2]pseudorotaxanes.

Short-range, Long-range and Transition State Interactions in the Denatured State of ACBP from Residual Dipolar Couplings

Residual dipolar couplings in the denatured state of bovine acyl-coenzyme A binding protein (ACBP) oriented in strained polyacrylamide gels have been shown to be a sensitive, sequence-specific probe for residual secondary structure. Results supporting this were obtained by comparing residual dipolar couplings under different denaturing conditions. The data were analyzed using the program molecular fragment replacement (MFR), which demonstrated α-helix propensity in four isolated stretches along the protein backbone, and these coincide with the location of native helices. This is in full agreement with earlier findings based on secondary chemical shift values. Furthermore, N–H residual dipolar couplings provided direct evidence for the existence of native-like hydrophobic interactions in the acid-denatured state of ACBP at pH 2.3. It was shown that replacement of the hydrophobic side-chain of residue Ile27 with alanine in helix A2 leads to large decreases of residual dipolar couplings in residues that form helix A4 in the native state. It is suggested that the Ile to Ala mutation changes the probability for the formation of long-range interactions, which are present in the acid-denatured state of the wild-type protein. These long-range interactions are similar to those proposed to form in the transition state of folding of ACBP. Therefore, the application of residual dipolar couplings in combination with a comparative mutation study has demonstrated the presence of precursors to the folding transition state under acid-unfolding conditions.

Synergy between Conserved ABC Signature Ser Residues in P-glycoprotein Catalysis

Functional roles of the two ABC signature sequences ("LSGGQ") in the N- and C-terminal nucleotide binding domains of P-glycoprotein were studied by mutating the conserved Ser residues to Ala. The two single mutants (S528A; S1173A) each impaired ATPase activity mildly, and showed generally symmetrical effects on function, consistent with equivalent mechanistic roles of the two nucleotide sites. Synergy between the two mutations when combined was remarkable and resulted in strong catalytic impairment. The Ser residues are not involved significantly in MgATP- or MgADP-binding or in interdomain communication between catalytic sites and drug binding sites. Retention of product MgADP is not the cause of reduced turnover. Mutation of Ser to Ala reduced the strength of interaction with the chemical transition state specifically, as shown by vanadate-ADP and beryllium fluoride-ADP trapping experiments. Therefore, the two conserved ABC signature motif Ser residues of P-glycoprotein cooperatively accelerate ATP hydrolysis via chemical transition state interaction. Because the transition state complex is currently believed to form in the dimerized state of the nucleotide binding domains, one may also conclude that both Ser-OH are necessary for correct formation of the dimer state.

Quantification of Functional Group Interactions in Transition States

A new version of the double mutant cycle approach has been used for the evaluation of weak noncovalent interactions in transition states.

The role of amino-acid residues Q39 and E451 in the determination of substrate specificity of the Spodoptera frugiperda beta-glycosidase.

The design of beta-glycosidases with planed substrate specificity for biotechnological application has received little attention. This is mostly a consequence of the lack of data on the molecular basis of the beta-glycosidase specificity, namely data on the energy of the noncovalent interactions in the enzyme-transition state complex. In an attempt to fill this gap, site-directed mutagenesis and enzyme steady-state kinetic experiments with different substrates were conducted, using as model a digestive beta-glycosidase (glycoside hydrolase family 1) from Spodoptera frugiperda (Lepidoptera) (Sfbetagly50). The active site of this enzyme was modeled based on its sequence and on crystallographic data of similar enzymes. Energy of noncovalent interactions in transition state between Sfbetagly50 amino acids and glycone hydroxyls was determined. Sfbetagly50 residue E451 seems to be a key residue in determining beta-glycosidase preference for glucosides vs. galactosides based on the following data: (a) The energy of the noncovalent interaction between glycone equatorial hydroxyl 4 with E451 in the transition state is about 60% higher than its interaction with Q39. (b) The energy of the E451-hydroxyl 4 interaction decreases more than the Q39-hydroxyl 4 interaction when hydroxyl 4 is changed from equatorial to axial position. (c) A Sfbetagly50 mutant, where E451 was substituted by A, hydrolyzes galactosides faster than glucosides. It was also shown that glycone hydroxyl 6 interacts favorably with Q39, but not with E451, probably due to steric hindrance. These interactions result in the beta-glycosidase hydrolyzing fucosides (6-deoxygalactosides) faster than glucosides and galactosides.

Metal ion catalyzed hydrolysis of ethyl p-nitrophenyl phosphate

15N isotope effects in the nitro group and 18O isotope effects in the phenolic oxygen have been measured for the hydrolysis of ethyl p-nitrophenyl phosphate catalyzed by several metal ions. Co(III)–cyclen at pH 7, 50 °C, gave an 15N isotope effect of 0.12% and an 18O one of 2.23%, showing that P–O cleavage is rate limiting and the bond is ∼50% broken in the transition state. The active catalyst is a dimer and the substrate is presumably coordinated to the open site of one Co(III), and is attacked by hydroxide coordinated to the other Co(III). Co(III)–tacn under the same conditions shows a similar 15N isotope effect (0.13%), but a smaller 18O one (0.8%). Zn(II)–cyclen at pH 8.5, 80 °C, gave an 15N isotope effect of 0.05% and an 18O one of 0.95%, suggesting an earlier transition state. The catalyst in this case is monomeric, and thus the substrate is coordinated to one position and attacked by a cis-coordinated hydroxide. Eu(III) at pH 6.5, 50 °C, shows a very large 15N isotope effect of 0.34% and a 1.6% 18O isotope effect. The large 15N isotope effect argues for a late transition state or Eu(III) interaction with the nitro group, and was also seen in Eu(III)-catalyzed hydrolysis of p-nitrophenyl phosphate [Bioorg. Chem. 28 (2000) 283].

Solvent and structural effects on the oxidation of 2,6‐diphenyl‐piperidin‐4‐ones by quinolinium chlorochromate

AbstractThe kinetics of oxidation of 3‐R‐2,6‐diphenyl‐piperidin‐4‐ones (where R = H, Me, Et, and i‐Pr) by quinolinium chlorochromate has been studied under pseudo‐first‐order conditions in different pure (protic and aprotic) solvents. The rate data is correlated with different solvent parameters using linear multiple regression analysis. From the regression coefficients, information on the solvent–reactant and the solvent–transition state interactions is obtained and the solvation models are proposed. Reasons for the difference in reactivity with structure are also discussed. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 585–588, 2002

Oxidative addition of Pd to C–H, C–C and C–Cl bonds: Importance of relativistic effects in DFT calculations

To assess the importance of relativistic effects for the quantum chemical description of oxidative addition reactions of palladium to C–H, C–C and C–Cl bonds, we have carried out a systematic study of the corresponding reactions of CH4, C2H6 and CH3Cl with Pd-d10 using nonrelativistic (NR), quasirelativistic (QR), and zeroth-order regularly approximated (ZORA) relativistic density functional theory (DFT) at the BP86/TZ(2)P level. Relativistic effects are important according to both QR and ZORA, the former yielding similar but somewhat more pronounced effects than the latter, more reliable method: activation barriers are reduced by 6–14 kcal/mol and reaction enthalpies become 15–20 kcal/mol more exothermic if one goes from NR to ZORA. This yields, for example, 298 K activation enthalpies ΔH298≠ of −5.0 (C–H), 9.6 (C–C) and −6.0 kcal/mol (C–Cl) relative to the separate reactants at ZORA-BP86/TZ(2)P. In accordance with gas-phase experiments on reactions of Pd with alkanes, we find reaction profiles with pronounced potential wells for reactant complexes (collisionally stabilized and observed in experiments for alkanes larger than CH4) at −11.4 (CH4), −11.6 (C2H6) and −15.6 kcal/mol (CH3Cl) relative to separated reactants [ZORA-BP86/TZ(2)P]. Furthermore, we analyze the height of and the relativistic effects on the activation energies ΔE≠ in terms of the activation strain ΔEstrain≠ of and the transition-state interaction ΔEint≠ between the reactants in the activated complex, with ΔE≠=ΔEstrain≠+ΔEint≠.

The tetrahymena ribozyme cleaves a 5'-methylene phosphonate monoester approximately 10(2)-fold faster than a normal phosphate diester: implications for enzyme catalysis of phosphoryl transfer reactions.

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 monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.