Methylene Phosphonate Monoester ∼102-fold Faster Than a Normal

© Copyright 2001 by the American Chemical Society

Volume 40, Number 37

September 18, 2001

Articles 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† Xiangmin Liao,‡,§ P. S. R. Anjaneyulu,‡ Jessica F. Curley,‡ Michael Hsu,‡ Markus Boehringer,⊥,| Marvin H. Caruthers,⊥ and Joseph A. Piccirilli*,‡ Howard Hughes Medical Institute, Departments of Biochemistry and Molecular Biology and of Chemistry, The UniVersity of Chicago, 5841 South Maryland AVenue, MC 1028, Chicago, Illinois 60637, and Departments of Chemistry and Biochemistry, UniVersity of Colorado at Boulder, Boulder, Colorado 80309 ReceiVed April 19, 2001; ReVised Manuscript ReceiVed June 28, 2001 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 dCCCUCUTmpTA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts ∼103-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 ∼10-fold and stimulates the chemical cleavage by ∼102-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 ∼102-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 10-fold excess of substrate and were carried out at 30 or 50 °C in a 10 µL volume containing ∼0.5 nM 5′-32P-labeled substrate, 50 mM NaMES (pH 5.5 or 7.0), 10 mM MgCl2, and G (or pG) (28). The same conditions were used for the site specific hydrolysis reaction, except that G (or pG) was omitted. Reaction conditions used to determine specific kinetic parameters are given in the footnotes of the tables. Reaction mixtures were preincubated without substrates at 50 °C for 30 min to renature the ribozyme. Substrate was incubated separately for 5 min at the temperature of the reaction. For reactions carried out at 30 °C, the preincubated ribozyme solution was transferred to a 30 °C water bath and incubated for 5 min before addition of substrate (31). Aliquots (1-2 µL) were withdrawn at specified times and the reactions quenched by dilution into 2 volumes of stop buffer (7.5 M urea, 20 mM EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue) followed by immediate cooling on dry ice. Substrates and products were separated by denaturing 20% PAGE. The fraction of substrate remaining at each time point [St/(St + Pt), where St and Pt are the quantities of substrate and product, respectively, at a specific time t] was quantitated using a PhosphorImager (Molecular Dynamics). Plots of ln[St/(St + Pt)] versus time were usually linear for approximately three half-lives and typically gave end points of g90%. Due to slow cleavage of dSTT and dSUA, rate constants for these reactions were measured from the initial rates. Reactions of dSTT, dSUA, dSTmpT, dSTOCH3, and dSTCH2CH3. Reactions were carried out at 30 °C, with ∼1 nM 5′-32P-labeled substrates, 50 mM NaMES (pH 7.0), 10 mM MgCl2, and 2 mM G using ribozyme concentrations of 10-1000 nM. Reactions of DNA-RNA Chimeric Substrates cSTT, cSTmpT, cSTOCH3, and cSTCH2CH3. Reactions were carried out at 50 °C, with ∼1 nM 5′-32P-labeled substrates, 50 mM NaMES (pH 7.0 or 5.5 as indicated), and 10 mM MgCl2, with or without

Liao et al. Scheme 2

G (or pG) as indicated. At 50 °C and pH 7.0, the rates of cleavage of cSTmpT at high ribozyme concentrations (g100 nM) in the presence of saturating G (or pG) were too fast to measure accurately; thus, only the second-order rate constants (kcat/Km)S and (kcat/Km)G were measured under these conditions. IGS′ Duplex Stability. To determine the affinity of binding of cSTT and cSTmpT to the IGS′ oligonucleotide (5′GGAGGGA), we used the substrate inhibition assay described previously (4, 31). Reactions were carried out at 50 °C, with ∼1 nM 5′-32P-labeled substrates, 50 mM NaMES (pH 7.0), 10 mM MgCl2, and 5 mM pG with 2 mM G using a ribozyme concentration of 100 (for cSTT) or 0.5 nM (for cSTmpT). Concentrations of the ribozyme were chosen to ensure that reactions were under kcat/Km conditions. The concentration of IGS′ was varied from 0 to 100 µM. Synthesis of Model Compounds. See the Supporting Information. Kinetics of Reactions of MNP and ENP with Nucleophiles. Reactions of MNP and ENP at 25 °C were monitored spectrophotometrically (400 nm) by following the formation of 4-nitrophenoxide (Scheme 2). Reaction mixtures contained 0.1 mM substrate (triethylammonium salt of MNP or ENP) for reactions with hydroxide and ethanolamine, and 10 mM substrate for reactions with hydroxylamine. For hydroxylamine reactions, aliquots were removed at various times and diluted 10-fold with aqueous KOH before the absorbance at 400 nm was measured. For reactions with hydroxide, the ionic strength was maintained at 1.7 by addition of KCl, and the concentration of potassium hydroxide was varied from 0.3 to 1.7 M. Reactions with ethanolamine and hydroxylamine were carried out at pH 9.7 and 6.0, respectively, in the corresponding hydrochloride buffer (0.1-2 M); the ionic strength was maintained at 2.0 by addition of KCl. For each reaction, more than 10 time points were monitored. To determine end points, the reactions with 1.7 M hydroxide were monitored until the absorbance at 400 nm stopped increasing, ∼350 h for MNP and ∼180 h for ENP. The final absorbance (A∞) for the reaction of each ester with hydroxide was used as the end point for all other reactions, including those in which ethanolamine or hydroxylamine was the nucleophile. Observed rate constants were determined from the slopes of plots of ln[(A∞ - At)/A∞] versus time, where At is the absorbance at time t. Initial rates were used to determine the rate constants for the ethanolamine and hydroxylamine reactions. All reactions were first-order with time, and the second-order rate constants in Table 5 were obtained from plots of the observed rate constants versus nucleophile concentration. The second-order rate constant for the reaction of MNP with hydroxide (Table 5) is in good agreement with that reported by Rahil and Pratt [5.82 × 10-5 M-1 min-1 (32)].

Ribozyme Cleavage of a 5′-Methylene Phosphonate Monoester

Biochemistry, Vol. 40, No. 37, 2001 10915

FIGURE 2: Endonuclease reaction catalyzed by the L-21 ScaI ribozyme (E). (A) The pathway for the reaction of the oligonucleotide substrate (CCCUCUAAAAA) with saturating guanosine (G) is shown. Binding of S is a two-step process (36-38). S first forms the open complex (EG‚S)o by base pairing with the internal guide sequence (IGS, GGAGGG) of the ribozyme (E) to give the P1 duplex. The P1 duplex then docks into the catalytic core via tertiary interactions to form the closed complex (EG‚S)c (4, 31, 36, 38-47). Four of the 2′-OH groups involved in tertiary stabilization [located at G22 and G25 of the IGS (43) and at C-2 and U-3 of the substrate] are represented by -OH on is the equilibrium constant for the P1 duplex. The kinetic and thermodynamic parameters have been defined previously (28, 30). KIGS d dissociation of S from (EG‚S)o. Ktertiary is the equilibrium constant for docking into the tertiary interactions to form the closed complex. The chemical cleavage step (kc) involves nucleophilic attack at the scissile phosphate diester by the 3′-hydroxyl group of a bound guanosine nucleoside (G). (B) The chemical step rate constant (kc) and the equilibrium constant for dissociation of S from EG‚S (KSd ) reflect different elementary steps for RNA and DNA substrates (4, 28, 30, 36, 39). For the DNA substrate, S binds in the open complex and, thus, KSd ) KIGS d , while the rate constant kc represents both the docking and chemical steps (4, 30, 38, 39). For substrates containing 2′-OH groups at C-2 and U-3, S binds in the closed complex and thus KSd represents both duplex-forming (KIGS d ) and docking (Ktertiary) steps, while the rate constant kc represents the chemical cleavage step (4, 36, 38, 39, 51).

Alkaline hydrolysis of MNP also was monitored by 1H NMR (Brucker DMX 500 spectrometer, 500 MHz). Reaction conditions were the same as those used for UV measurements, except that the MNP concentration was 10 mM and the solvent was D2O. Reactions were followed by integration of the O-methyl resonance of the starting diester (doublet at δ 3.73 relative to water) and that of the product monoester, methyl phosphate (doublet at δ 3.12). 1H NMR showed that 4-nitrophenolate anion and methyl phosphate were the only products formed in the reaction. After alkaline hydrolysis was completed, 31P NMR showed that the single 31P resonance of the starting material (δ 3.18 relative to aqueous phosphoric acid) was completely converted to a single resonance (δ 5.66) of the methyl phosphate product. Plots of ln[St/(St + Pt)] versus time were linear for more than three half-lives. The second-order rate constant for the reaction of MNP with hydroxide obtained by NMR was in good agreement with that obtained by UV spectroscopy (Table 5). Some nucleophiles, including hydroxide, react with phenyl phosphate esters or phenyl phosphonate esters via two pathways: (1) SN2(P) attack at phosphorus or (2) nucleophilic aromatic substitution at C1 of the phenyl ring (2, 33). The observed rates of the reaction of MNP and ENP with nucleophiles could therefore represent the sum of the rates for both pathways. Because the rate for attack of nucleophiles at phosphorus is of interest for this work, it is necessary to determine the contribution of each pathway to the overall rate of reaction. 31P NMR product analysis (33) of reactions with [18O]hydroxide ion shows that the ratio of [16O]methyl

phosphate to [18O]methyl phosphate or [16O]ethyl phosphonate to [18O]ethyl phosphonate is not detectably different from the ratio of 16O to 18O in the solvent. These results indicate that reactions of both MNP and ENP with hydroxide occur predominantly through attack at phosphorus. To address the contribution by the amine nucleophiles to each pathway, we monitored the reactions of 2 M ethanolamine (pH 9.7) or hydroxylamine (pH 6.0) with 10 mM MNP by 1 H NMR over a period of 167 days. During this period of time, >60% of the MNP reacts with ethanolamine, and >90% with hydroxylamine. 1H NMR showed only the formation of p-nitrophenol; we could detect no p-nitrophenylamines, which would result from attack at carbon. Thus, the second-order rate constants for the reactions of the amine nucleophiles represent attack at phosphorus. This is consistent with the observation that a variety of amine nucleophiles show little or no tendency to react at the aromatic carbon of p-nitrophenyl methyl phosphonate (1). RESULTS The L-21 ScaI ribozyme, derived from the Tetrahymena thermophila self-splicing group I intron, catalyzes phosphoryl transfer between RNA or DNA substrates and guanosine (or one of its 5′-phosphorylated forms) in a reaction analogous to the first step of self-splicing (28, 30, 34, 35). -5 -3 -1

+2

E

CC CU CU AA AAA + G 98 CCCUCU + GAAAAA (1) Water or hydroxide can replace G as the nucleophile in an

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FIGURE 3: Tetrahymena ribozyme cleaves a DNA substrate containing a methylene phosphonate linkage at the scissile phosphorus center much faster than the natural phosphate diester linkage. (A) Structures of the DNA substrates used in this work (left, dSTmpT; center, dSTT; right, dSUA). The deoxyribose phosphate backbone at the cleavage site is shown to illustrate the chemical nature of the single-atom change. The “normal” substrates have phosphate diester linkages at all backbone positions, and the “mp” substrate has the 5′-methylene phosphonate monoester linkage at the cleavage site and phosphate diester linkages at all other sites. (B) Time course and product characterization for ribozyme reactions of the substrates shown in panel A. Reactions were carried out at 30 °C with 1000 nM E, 2 mM G, ∼1 nM 5′-32Plabeled DNA substrate as indicated, 50 mM NaMES (pH 7.0), and 10 mM MgCl2. The products were identified as P (dCCCUCT for ribozyme cleavage of dCCCUCTmpTAAAA and dCCCUCTTAAAA and dCCCUCU for ribozyme cleavage of dCCCUCUAAAAA) by comparison with the ladders generated by partial digestion with S1 nuclease (lanes denoted by S1).

analogous sequence specific hydrolysis reaction (28). The binding of oligonucleotide substrate (S) to the ribozyme (E) involves two steps (Figure 2) (36-38). First, S interacts with E by complementary Watson-Crick base pairing with the internal guide sequence (IGS) to form an open E‚S complex (E‚S)o; then, the resulting P1 duplex docks into the ribozyme active core via tertiary interactions to form the closed complex (E‚S)c (4, 31, 36, 38-47). Quantitative analysis of the reaction rate and equilibrium constants involved in this endonuclease reaction pathway has provided a kinetic and thermodynamic framework for understanding the energetics of catalysis and for interpreting the results of mutagenesis experiments (28-30, 48-51). The Ribozyme CleaVes a Phosphonate Monoester ∼103Fold Faster Than a Phosphate Diester. We synthesized an all-DNA substrate dCCCUCTmpTA4 (dSTmpT, Figure 3A) containing at the cleavage site two thymidine residues linked by a methylene phosphonate monoester. We used thymidine residues in place of the natural UA residues because of the availability of the 3′-phosphoramidite of the 5′-deoxy-5′methylenephosphonate-linked thymidine dimer (22). Sub-

stitution of dT for dU at the cleavage site previously was shown to have little effect on the cleavage rate of normal DNA substrates (28, 44). We also synthesized two control substrates: dSTT, containing two thymidine residues linked by a normal phosphate diester, and dSUA, the standard DNA substrate (Figure 3A). When the 5′-32P-end-labeled substrates were incubated at 30 °C (pH 7.0) in the presence of 2 mM G and 1 µM L-21 ScaI ribozyme, the modified substrate dSTmpT reacted almost completely within 1 h (t1/2 ∼ 0.5 h; Figure 3B, lanes 3-7), whereas the substrates containing the normal phosphate linkage, dSTT and dSUA, reacted much more slowly (t1/2 ∼ 640 h, lanes 10-14; and t1/2 ∼ 83 h, lanes 17-21). The product of ribozyme cleavage of dSTmpT comigrated with that from dSTT and with the appropriate product (*pdCCCUCT) from S1 nuclease digestion of dSTmpT (lane 2) and dSTT (lane 9), confirming that the ribozyme cleaves the substrate at the methylene phosphonate linkage. We measured the dependence of kobsd on the concentration of E for dSTmpT, dSTT, and dSUA and fit the data by nonlinear least-squares analysis to determine the kinetic parameters kc and Km (Table 1). For the standard DNA substrate, dSUA,

Ribozyme Cleavage of a 5′-Methylene Phosphonate Monoester Table 1: Comparison of DNA Substrates Containing a Normal Phosphate Linkage or a 5′-Deoxy-5′-methylene Phosphonate Linkage at the Cleavage Sitea,b substrate dSUA dC3UCUA5 dSTT dC3UCTTA4 dSTmpT dC3UCTmpTA4

kcat/Kmc kc (×105 d (M-1 min-1) min-1) 1543 223 280000

14 1.8 2600

e krel c

8 (1) 1400

Kd f g (nM) Krel d 68 43 61

1.6 (1) 1.4

a Reactions with 0-2000 nM ribozyme, 2 mM G, and ∼1 nM 5′end-labeled substrate in 50 mM NaMES (pH 7.0 and 25 °C) and 10 mM MgCl2 at 30 °C. G was shown to be near saturating for dS with a Km(G) of ∼1 mM (49), and no correction for saturation was made. b The given values represent nonlinear least-squares fits to the data. c The second-order rate constant for the reaction of EG and substrate (30). d The rate constant kc is that for the chemical conversion of the dS dSTT (EG‚S)o ternary complex (39). e krel , representing the rate c ) kc /kc constant for cleavage of dS relative to that of dC3UCTTA4. f The equilibrium constant Kd for dissociation of S from the EG‚S complex. The values were obtained from nonlinear least-squares fits to plots of dS dSTT kobsd vs EG, assuming Km ) Kd. g Krel d ) Kd /Kd , representing the dissociation constant for dS relative to that of dC3UCTTA4.

Km equals Kd, the equilibrium constant for dissociation of S from the open complex, (EG‚S)o / EG + S (Figure 2B), and should therefore reflect the stability of the P1 duplex under the reaction conditions (4, 30, 38, 52-54). The rate constant kc represents both the docking and chemical steps. The values of Kd and kc for dSTmpT, dSTT, and dSUA show that the unexpectedly fast cleavage rate of the methylene phosphonate linkage manifests itself only in kc and not in Kd. The similar Kd values for the three substrates (within error) indicate that the substrates bind in the open complex with equal affinity (Table 1). Thus, the 5′-O to 5′-CH2 substitution has little effect on the stability of the P1 duplex, consistent with the previous observation that a 5′-CH2 modification does not affect oligonucleotide duplex stability (26). In contrast, the 5′-CH2 modification in dSTmpT increased kc by (1.4 × 103)-fold relative to that of the control substrate dSTT (Table 1), indicating 4.4 kcal mol-1 of transition state stabilization from the 5′-CH2 substitution. Because kc for DNA substrates reflects both the docking and chemical steps, the larger kc value may arise from more favorable docking of the substrate into the closed complex and/or from faster chemistry. The 8-fold smaller value of kc for dSTT compared to that for dSUA (Table 1) was unexpected because previous work had shown that substitution of the deoxyuridine immediately preceding the cleavage site with thymidine (dSTA) has no effect on activity (30) and because the adenine base of the residue immediately following the cleavage site (+1) putatively makes no tertiary interactions (39, 52). We confirmed that dSTA reacts as efficiently (within 40%) as dSUA but found that dSUT, in which the adenosine immediately following the cleavage site was substituted with thymidine, reacts ∼2.5fold slower than dSUA. Thus, the 8-fold lower reactivity of dSTT compared to dSUA appears to arise from the combination of T’s, but we do not know the reason for this effect (see below). The modified postsynthetic workup involving treatment with 2-nitrobenzaldoxime and tetramethylguanidine (see Materials and Methods) does not cause the lower reactivity of dSTT, as dSTT prepared without this modified workup still reacts 8-fold slower than dSUA. To account for the strikingly favorable effect of the 5′-O to 5′-CH2 substitution, we analyzed further the effect of this

Biochemistry, Vol. 40, No. 37, 2001 10917 atomic perturbation in three ways. (1) We incorporated 2′hydroxyl groups into the substrates so that we could observe differences in docking. (2) We studied the effect of the 5′-O to 5′-CH2 substitution in the context of substrates in which the 3′-terminal residues (+1 to +5) were replaced with a methoxy (-OCH3) and an ethyl group (-CH2CH3), respectively (Scheme 1). (3) We measured the intrinsic differences in the reactivity of a model phosphonate monoester and a phosphate diester toward nucleophiles. Reactions with Chimeric DNA-RNA Substrates. The 2′hydroxyl groups at positions U-3 and C-2 stabilize docking of the P1 duplex into the closed complex by factors of 65 and 18, respectively, and maintain this stabilization in the transition state for the chemical step (4, 39, 40). To determine whether these 2′-hydroxyl groups have a similar stabilizing effect on the cleavage reaction of the methylene phosphonate substrate and to attribute the faster cleavage rate of the methylene phosphonate substrate to effects on the docking versus the chemical step, we synthesized substrates containing 2′-hydroxyl groups at U-3 and C-2 (cSrUT, cSTT, and cSTmpT). Because the methylene phosphonate linkage had not been incorporated previously into an oligonucleotide containing ribose residues, we analyzed cSTmpT by partial alkaline cleavage and S1 nuclease digestion (Figure 4). The ladders generated for cSTmpT (lane 7) were similar to those for cSTT (lane 11) and cSrUT (lane 3). Additionally, the product that resulted from cleavage by the L-21 ScaI ribozyme (lane 8), presumably *pd(CCC)r(UC)dT, comigrated with the product from the cleavage of cSTT (lane 12). Ribozyme reactions were carried out at 50 °C so that we could determine P1 duplex stability under the same conditions using the method of substrate inhibition (4, 31). As expected, the second-order rate constants for reactions at 50 °C and pH 7.0 of cSTT and cSTmpT with EG (Table 2) increased by ∼2 orders of magnitude compared to those for their alldeoxy counterparts (dSTT and dSTmpT, respectively), indicating that the 2′-hydroxyl groups at U-3 and C-2 contribute energetic stabilization in the transition state. For substrates lacking the 2′-hydroxyl at position -1, such as cSTT, the rate-limiting step for (kcat/Km)S is chemical cleavage (55) so that (kcat/Km)S reflects the overall free energy difference for the reaction EG + S f products. Under these conditions, cSTmpT reacts ∼400-fold faster than cSTT (data not shown). However, this comparison of (kcat/Km)S values for cSTT and cSTmpT could be misleading because different steps could be rate-limiting for each substrate (see below). Determining the Effect of the 5′-O to 5′-CH2 Substitution on the Energetics of Binding. The equilibrium dissociation constant for binding of S to EG (KSd ) equals Km for S in the reaction with the EG complex if S dissociates from the EG‚S complex much faster than the ternary complex (EG‚S) reacts to give products, i.e., koff . kc. The reaction of cSTT likely satisfies this condition because this substrate reacts very slowly (Table 2). However, cSTmpT reacts with a (kcat/Km)S value (6.2 × 107 M-1 min-1; Table 2) strikingly similar to the value of 108 M-1 min-1 for duplex formation (28, 36, 51), indicating that the 5′-CH2 substitution might have accelerated the chemical step to the extent that binding of the oligonucleotide substrate to the IGS becomes ratelimiting. Therefore, Km for cSTmpT may not equal Kd, and the kinetic parameters for cSTmpT and cSTT should not be

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FIGURE 4: Ribozyme cleavage and characterization of chimeric DNA-RNA substrates d(CCC)r(UCU)Tr(AAAA) (cSrUT), d(CCC)r(UC)TmpTr(AAAA) (cSTmpT), and d(CCC)r(UC)TTr(AAAA) (cSTT). Lanes are as follows: input, untreated 5′-32P-labeled chimeric substrate; HO-, partial alkaline hydrolysis of S obtained by treatment with 0.1 M NaHCO3 (95 °C for 40 min); S1, partial digestion by S1 nuclease; L-21, ribozyme cleavage reaction after 1 min (cSrUT and cSTmpT) or 4 h (cSTT). Ribozyme reactions were single-turnover reactions whose mixtures contained