Theoretical and Experimental Considerations on the Hammerhead Ribozyme Reactions: Divalent Magnesium Ion Mediated Cleavage of Phosphorus-Oxygen Bonds

Uebayasi, Masami; Koguma, Tetsuhiko; Sawata, Shinya Y.; Shimayama, Takashi; Taira, Kazunari; Uchimaru, Tadafumi

doi:10.1021/jo00103a038

Cited by 49 publications

(97 citation statements)

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“…Several lines of evidence are consistent with a double-metal-ion mechanism of catalysis ( Fig. 1A), in which two Mg 2 ions are coordinated directly with the attacking and the leaving oxygens (Taira et al 1990;Steitz & Steitz 1993;Uchimaru et al 1993;Uebayashi et al 1994;Sawata et al 1995;Kumar et al 1996;Pontius et al 1997;Zhou et al 1997;Lott et al 1998;Zhou & Taira 1998;Yoshinari & Taira 2000).…”

Section: Introductionmentioning

confidence: 54%

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

et al. 2000

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Background: Several reports have appeared recently of experimental evidence for a double-metal-ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes. In one case, hammerhead ribozyme-mediated cleavage was analysed as a function of the concentration of La 3 ions in the presence of a ®xed concentration of Mg 2 ions so that the role of metal ions that are directly involved in the cleavage reaction could be monitored. The resultant bell-shaped curve for activation of cleavage was used to support the proposed doublemetal-ion mechanism of catalysis. However, other studies have demonstrated that the binding of a metal ion (the most conserved P9 metal ion) to the pro-Rp oxygen (P9 oxygen) of the phosphate moiety of nucleotide A 9 and to the N7 of nucleotide G 10.1 is critical for ef®cient catalysis, despite the large distance (<20 A Ê ) between the P9 metal ion and the labile phosphodiester group in the ground state. In fact, it was demonstrated that an added Cd 2 ion binds ®rst to the pro-Rp phosphoryl P9 oxygen but not with the pro-Rp phosphoryl oxygen at the cleavage site.

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Section: Introductionmentioning

confidence: 54%

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

et al. 2000

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“…In contrast, ab initio calculations predict that the bond breaking portion of the cleavage reaction should provide the largest contribution to the activation energy barrier in hammerhead catalysis (19). Stabilization of the leaving group (the 5Ј-oxygen) with a metal cation would therefore be expected to lower the activation barrier and increase the cleavage rate.…”

Section: Results and Interpretationsmentioning

confidence: 98%

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Pontius

Lott

Hippel

1997

Proc. Natl. Acad. Sci. U.S.A.

121

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Significant cleavage by hammerhead ribozymes requires activation by divalent metal ions. Several models have been proposed to account for the inf luence of metal ions on hammerhead activity. A number of recent papers have presented data that have been interpreted as supporting a one-metal-hydroxide-ion mechanism. In addition, a solvent deuterium isotope effect has been taken as evidence against a proton transfer in the rate-limiting step of the cleavage reaction. We propose that these data are more easily explained by a two-metal-ion mechanism that does not involve a metal hydroxide, but does involve a proton transfer in the rate-limiting step.Hammerhead ribozymes are one of several types of RNA enzymes (1). The term hammerhead refers to a small domain, found in some viral satellite RNAs, that is required for RNA self-cleavage (2). This small domain is necessary for cleavage activity on RNA sequences that form complementary base pairs with the hammerhead (3). Crystal structures have been obtained for hammerhead ribozymes complexed with different noncleavable substrate analogues (4, 5). Because of its small size and simple structure, the hammerhead has been extensively studied as a prototypic RNA enzyme. Fig. 1 depicts a hammerhead ribozyme that is base paired to a 17-nucleotide substrate RNA. This hammerhead, called HH16 (6), has 8 bp in both recognition helices that flank the cleavage site on the substrate. Cleavage occurs on the 3Ј side of a cytosine residue in the substrate strand.Several models have been proposed for the cleavage of RNA phosphodiester bonds by hammerhead ribozymes; most models include a solvated metal hydroxide ion at the catalytic center (4,7,8). In these metal-hydroxide-ion models ( Fig. 2A) a divalent metal hydroxide binds to the pro-R oxygen on the phosphate at the site of cleavage and activates the ribozyme primarily by removing a proton from the 2Ј-OH of the attacking nucleotide residue. The activated 2Ј-O Ϫ species then acts as the nucleophile by attacking the phosphodiester bond.A two-metal-ion model has also been proposed for the hammerhead ribozyme (9). This model is based on mechanisms that have been put forward to explain the activities of alkaline phosphatase and the 3Ј-5Ј exonuclease of DNA polmerase I (10, 11). Similar two-metal-ion models have been proposed for a wide variety of protein and RNA enzymes that mediate phosphoryl transfer reactions (9). In one such model for the hammerhead ribozyme (Fig. 2B), both divalent metal ions bind to the pro-R oxygen on the phosphate at the site of cleavage. The first metal ion (in site A) interacts directly with the 2Ј-oxygen of the attacking ribonucleotide residue, while the second metal ion (in site B) interacts with the 5Ј-oxygen leaving group. In a two-metal-ion mechanism, the metal ion in site A is thought to facilitate the formation of the 2Ј-oxyanion that acts as the nucleophile, and the metal ion in site B is considered to stabilize the oxyanion leaving group (11). Both metal ions may also stabilize the specific geometry ...

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“…The ribozyme and its corresponding substrate were synthesized on an ABI DNA/RNA synthesizer (model 392; Applied Biosystems, Foster City, CA) and purified by HPLC and electrophoresis in a polyacrylamide gel as described previously [13,16,17]. RNA reagents were purchased from American Bionetics Inc. (ABN; Hayward, CA).…”

Section: Synthesis Of the Ribozyme And Its Substratementioning

confidence: 99%

“…These conditions were achieved by employing high concentrations of either the ribozyme (<3.8/.tM) or the substrate (< 1.1 ¢tM). The Km value of the ribozyme for its substrate was 0.02 ¢tM at 37°C under the present conditions [13,16,17].Reactions were stopped by removal of aliquots from the reaction mixture at appropriate intervals and mixing them with an equal volume of a solution of 100 mM EDTA, 9 M urea, 0.1% xylene cyanol, and 0.1% Bromophenol blue. Substrates and 5'-cleaved products were separated by electrophoresis on a 20% polyacrylamide/7 M urea denaturing gel and were detected by autoradiography.…”

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“…First, the substrate (and Mg 2÷ ions) binds to the ri~ozyme to form a Michaelis-Menten complex via formation of ~ase pairs with stems I and III (k ..... ). Then, a specific :ghosphodiester bond in the bound substrate is cleaved by the lction of Mg 2+ ions (k~eav; the ribozyme is recognized to funcion as a metalloenzyme [5,[8][9][10][11][12][13][14]). This cleavage produces prodJcts with 2',3'-cyclic phosphate and 5'-hydroxyl groups.…”

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Temperature‐dependent change in the rate‐determining step in a reaction catalyzed by a hammerhead ribozyme

Takagi

Taira

1995

FEBS Letters

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To characterize the reaction catalyzed by a hammerhead ribozyme, the dependence on temperature of the reaction was examined. An Arrhenius plot revealed a transition that indicated a temperature-dependent change in the activation energy at around 25°C. Thermodynamic parameters oftbe reaction were estimated at 10 and 35°C. The analyses led to the following conclusions. At 25--50°C, the chemical cleavage step (k~a,) was the rate-determining step, and the cleaved fragments dissociated from the ribozyme at a higher rate than the rate of the chemical reaction. When the temperature was below 25°C, the cleaved fragments adhered to the ribozyme more tightly and the product dissociation step became the rate-determining step. Above 50 ° C, the rate of the reaction decreased because, at such high temperatures, the formation of the Michaelis-Menten complex (duplex formation) was hampered by thermal melting. A conformational change in the ribozyme-substrate complex was not the ratedetermining step at any of the temperatures examined. i,;ey words: Ribozyme; Hammerhead; Arrhenius plot; Kinetics; thermodynamics IntroductionThe hammerhead ribozyme is one of the smallest RNA en-/~ymes [1 3]. Because of its small size and potential utility as an antiviral agent, it has been extensively investigated in terms of ~he mechanism of its action and possible applications in vivo : 1-7]. In naturally occurring hammerhead ribozymes, reactions tre catalyzed in cis (intramolecularly), with the target and catdytic strands being part of a single RNA molecule. The trans-,tcting hammerhead ribozyme developed by Haseloff and Gerach [3] consists of an antisense section (stems I and III) and ~t catalytic domain with a flanking stem II/loop section ( Fig. 1). Fhe minimum reaction scheme can be described as shown in !Zig. 2. First, the substrate (and Mg 2÷ ions) binds to the ri~ozyme to form a Michaelis-Menten complex via formation of ~ase pairs with stems I and III (k ..... ). Then, a specific :ghosphodiester bond in the bound substrate is cleaved by the lction of Mg 2+ ions (k~eav; the ribozyme is recognized to funcion as a metalloenzyme [5,[8][9][10][11][12][13][14]). This cleavage produces prodJcts with 2',3'-cyclic phosphate and 5'-hydroxyl groups. Firally, the cleaved fragments dissociate from the ribozyme and the liberated ribozyme is now available for a new series of catalytic events (k~is~).In the reactions catalyzed by many protein enzymes, the kinetics are not as simple as those described above since, in many cases, a conformational change can become the ratedetermining step [15]. In this study, we examined the possibility of a rate-determining conformational change in a hammerhead ribozyme at low temperature by measuring the dependence on temperature of kcat. An Arrhenius plot revealed distinct changes in the rate-determining step. A combination of single-and multiple-turnover kinetics in the Arrhenius plot revealed that a conformational change was not the rate-determining step. Materials and methods Synthesis of the ribozyme and ...

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Theoretical and Experimental Considerations on the Hammerhead Ribozyme Reactions: Divalent Magnesium Ion Mediated Cleavage of Phosphorus-Oxygen Bonds

Cited by 49 publications

References 3 publications

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Temperature‐dependent change in the rate‐determining step in a reaction catalyzed by a hammerhead ribozyme

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Theoretical and Experimental Considerations on the Hammerhead Ribozyme Reactions: Divalent Magnesium Ion Mediated Cleavage of Phosphorus-Oxygen Bonds

Cited by 49 publications

References 3 publications

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Significant activity of a modified ribozyme with N7‐deazaguanine at G10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Observations on catalysis by hammerhead ribozymes are consistent with a two-divalent-metal-ion mechanism

Temperature‐dependent change in the rate‐determining step in a reaction catalyzed by a hammerhead ribozyme

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Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes

Significant activity of a modified ribozyme with N7‐deazaguanine at G_10.1: the double‐metal‐ion mechanism of catalysis in reactions catalysed by hammerhead ribozymes