Nucleic Acids Research, 2001, Vol. 29, No. 9 1815-1834
© 2001 Oxford University Press
Survey and Summary |
SURVEY AND SUMMARY
Recent advances in the elucidation of the mechanisms of action of ribozymes
1Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Science City 305-8562, Japan, 2Polish Academy of Science, Center of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 Lodz, Poland and 3Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan
Received as resubmission February 15, 2001; Revised and Accepted February 27, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme–substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pKa of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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Naturally existing catalytic RNAs include hammerhead, hairpin, hepatitis delta virus (HDV) and Varkud Satellite (VS) ribozymes; group I and II introns; and the RNA subunit of RNase P (1–6). The structures of these catalytic RNAs are shown in Figure 1. In addition, recent structural and chemical analyses strongly suggest that the ribosomal RNA is a ribozyme (7–10) and the possibility that the RNA component of the spliceosome might also be a ribozyme (11).
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Extensive efforts over the 15 years that followed the discovery of ribozymes (1,2) have revealed details of the mechanisms of the ribozyme-mediated cleavage (or ligation) of RNA. Ribozymes have been considered to be ‘fossil molecules’ that originated in a hypothetical prebiotic RNA world and it is likely that elucidation of their mechanisms of action will enhance our understanding of the life processes of primitive organisms (12–33). Since the earliest research on ribozymes, it was assumed that all ribozymes are metalloenzymes that require divalent metal ions for catalysis and that all must operate by a basically similar mechanism. However, recent advances have revealed examples of cleavage by hairpin ribozymes that are independent of divalent metal ions (34–39). Thus, the various types of ribozyme appear to exploit different cleavage mechanisms, which depend upon the architecture of the individual ribozyme. Furthermore, it was proposed recently that nucleobases in the HDV ribozyme might be candidates for participants in acid/base catalysis (40–42).
In addition, even hammerhead ribozymes, generally characterized as typical metalloenzymes, can no longer be unambiguously categorized (43,44). Recent findings indicate that the hammerhead ribozyme might operate via a variety of cleavage mechanisms, depending on the conditions of the reaction. Nevertheless, there is no doubt that RNA catalysts with groups that are poorly functional under physiological conditions do cooperate with metal ions to exert their catalytic activity and that many ribozymes can exploit divalent metal ions as cofactors and as stabilizers of their respective higher-order structures. The widespread potential utility of RNA molecules as catalysts and the events during reactions catalyzed by ribozymes, in particular the actions of catalytic functional groups such as metal ions and pKa-perturbed nucleobases, have generated considerable interest (1–49). In this article we shall review various naturally existing ribozymes that cleave RNAs, focusing mainly on the various mechanisms of catalysis.
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CLEAVAGE OF THE PHOSPHODIESTER BOND |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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For the cleavage of RNA phosphodiester linkages, three types of large ribozyme, namely, group I and II introns and the catalytic RNA subunit of RNase P, accept external nucleophiles (the 2'-OH group of an internal adenosine in the case of the group II intron). By contrast, small ribozymes, such as hammerheads, hairpins, HDV and the VS ribozyme, use an internal nucleophile, namely, the 2'-oxygen of the ribose moiety at the cleavage site, with resultant formation of a 3'-terminal 2',3'-cyclic phosphate. In general, ribozymes catalyze the endonucleolytic transesterification of the phosphodiester bond, requiring structural and/or catalytic divalent metal ions under physiological conditions. The reactions catalyzed by small ribozymes are considered to be roughly equivalent to the non-enzymatic hydrolysis of RNA, with inversion of the configuration at a phosphorus atom suggesting a direct in-line attack with development of a pentacoordinate transition state or intermediate. The chemical cleavage requires two events, which can occur either via a two-step mechanism or via a concerted mechanism (4,5,25,45).
In the first step of the non-enzymatic hydrolysis of RNA (25,50–52), the 2'-OH attacks the adjacent scissile phosphate, acting as an internal nucleophile (transition state 1; TS1) (Fig. 2). In the second step, the 5'-oxygen of the leaving nucleotide is released to produce a 3'-end 2',3'-cyclic phosphate and a 5'-OH terminus (transition state 2; TS2). Of the two putative transition states, TS2 is the overall rate-limiting state [i.e., attack by the 2'-OH on the phosphorus atom is easier than cleavage of the P-O(5') bond and, thus, TS2 always has higher energy than TS1] (25). This conclusion was confirmed in experiments with an RNA analog with a 5'-mercapto leaving group. If the formation of the intermediate were the rate-limiting step (i.e., if TS1 were a higher-energy state than TS2) in the natural RNA, the phosphorothiolate RNA (RNA with a 5'-bridging phosphorothiolate at the scissile linkage) should be hydrolyzed at a rate similar to the rate of the hydrolysis of the natural RNA because the 5'-bridging phosphorothiolate linkage would not be expected to enhance the attack by the 2'-oxygen (53). By contrast, if the decomposition of the intermediate were the rate-limiting step (i.e., if TS2 were a higher-energy state than TS1) in the natural RNA, the phosphorothiolate RNA would be expected to be hydrolyzed much more rapidly than the natural RNA because the pKa of a thiol is >5 units lower than that of the corresponding alcohol. Several groups have confirmed that the phosphorothiolate RNA is significantly more reactive than the corresponding natural RNA in non-enzymatic hydrolytic reactions (25,45,54–56) and, thus, TS2 is, indeed, always a higher-energy state than TS1.
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POSSIBLE CATALYTIC FUNCTIONS OF METAL IONS IN THE CLEAVAGE OF RNA |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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If ribozymes operate as metalloenzymes (4,5,15–25,27,45), the possible catalytic functions of metal ions can be summarized as follows (Fig. 3).
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· A metal-coordinated hydroxide ion might act as a general base, abstracting the proton from the 2'-OH (Fig. 3b) or, alternatively, a metal ion might act as a Lewis acid to accelerate the deprotonation of 2'-OH by coordinating directly with the 2'-oxygen (Fig. 3d).
· The developing negative charge on the 5'-oxygen leaving group might be stabilized by a proton that is provided by a solvent water molecule or by a metal-bound water molecule as a general acid catalyst (Fig. 3a) or, alternatively, by direct coordination of a metal ion that acts as a Lewis acid catalyst (Fig. 3c).
· Direct coordination of a metal ion to the non-bridging oxygen might render the phosphorus center more susceptible to nucleophilic attack (electrophilic catalysis; Fig. 3e) or, alternatively, hydrogen bonding between a metal-bound water molecule and the non-bridging oxygen might stabilize the charged trigonal-bipyramidal intermediate (or transition state).
Metal ions can function in several different ways as cofactors in ribozyme-catalyzed reactions, as described above, and proposed mechanisms for the reactions catalyzed by several ribozymes have taken advantage of such functions. Moreover, it is difficult to imagine that a specific ribozyme might exploit multiple mechanisms (for example, coexistence in a reaction of the left structure and the central structure in Figure 3 at the transition state of a ribozyme-catalyzed reaction) under a single set of physiological conditions. Significant aspects of these functions of metal ions might be subsumed by nucleobases if their pKa values could be adjusted appropriately. The full details of the mechanisms of action of metalloenzymes remain to be elucidated.
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LARGE RIBOZYMES |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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The group I intron, the group II intron and the RNA subunit of RNase P are categorized as large ribozymes. Group I and II introns are found in bacteria and in the organelles of higher plants, fungi and algae (57,58). These introns are spliced out of their primary transcripts by a two-step mechanism [Fig. 4A(i) and B(i)]. In the first step of splicing, the 5' splice site is attacked by the 3'-OH of the external guanosine (group I). Alternatively, it is attacked by the 2'-OH of the internal adenosine residue or by a hydroxide ion, in the case of hydrolysis (group II). In the second step, the 3'-OH of the 3'-end of the upstream exon attacks the 3' splice site to produce splicing products.
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RNase P is an endonuclease that generates the mature 5'-ends of tRNAs. In bacterial RNase P, the RNA subunit (RNase P ribozyme) has catalytic activity and the protein component is thought to act only to facilitate the binding of the anionic RNase P ribozyme to its substrate. However, mutations in either the gene for the RNA or the gene for the protein can inactivate RNase P in vivo, demonstrating that both components are necessary for natural enzymatic activity. In the cleavage by the RNase P ribozyme, a scissile-site phosphate is attacked by a hydroxide ion to leave a 3'-oxygen and to produce a 5'-phosphate terminus.
All of these ribozyme reactions proceed with inversion of configuration at a phosphorus atom (59–62), suggesting direct in-line attack with development of a pentacoordinate transition state or intermediate (59–72).
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THE MECHANISM OF REACTIONS CATALYZED BY THE GROUP I INTRON RIBOZYME |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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In studies of the reactions mediated by the ribozyme from the Tetrahymena group I intron, detailed kinetic and thermodynamic analysis, combined with modifications at the atomic level, helped to define the reaction mechanism of this ribozyme at the atomic level (18,63,66,68–72). Modification at the atomic level has generally involved replacement by a sulfur atom of an oxygen atom that has the potential to interact with a catalytically important metal ion. The observed reduction in the cleavage rate in the presence of Mg2+ ions after such modification (the ‘thio effect’) and the observed restoration of a normal cleavage rate in the presence of Mn2+ ions (the ‘manganese rescue effect’) have been taken as evidence that supports the direct coordination of the atom in question with a metal ion. This phenomenon can be explained by the HSAB (Hard and Soft, Acid and Base) rule (73,74). According to this rule, a ‘hard acid’, such as a Mg2+ ion, prefers to bind to a ‘hard base’ oxygen atom rather than to a ‘soft base’ sulfur atom. By contrast, a ‘soft acid’, such as Cd2+ or Zn2+ ions, prefers to bind to a ‘soft base’ sulfur atom. A Mn2+ ion is also softer than a Mg2+ ion and, thus, the former can bind to a soft sulfur atom (as well as to a hard oxygen atom). This ability of Mn2+ ions is believed to be the origin of the manganese rescue effect.
Analysis of both the thio effect and of soft acid rescue effects, such as the rescue effects of Cd2+, Mn2+ and Zn2+ ions, has contributed significantly to our understanding of the catalytic mechanism of the first step of the reaction catalyzed by the group I intron. Such analysis has revealed the importance of three to four independent metal ions, as shown in Figure 4A(ii) (69,71). It is generally accepted that the group I intron is a metalloenzyme that operates via the general double-metal-ion mechanism of catalysis, in which a Mg2+ ion at site (b) [see Fig. 4A(ii) for locations of (a), (b), (c) and (d)] enhances the deprotonation of the 3'-OH of the guanosine nucleophile and a Mg2+ ion at site (a) stabilizes the leaving 3'-bridging oxygen of U–1 in the transition state. In this case, the divalent metal ions function as Lewis acids for activation of the nucleophile and stabilization of the leaving group by coordinating directly with them (63). This mechanism corresponds, in the reactions catalyzed by small ribozymes, to the central mechanism shown in Figure 3 with the stabilization of both TS1 and TS2 (Fig. 2) by two metal ions.
These details of coordination at the catalytic site were derived from the following observations. The substitution of the 3'-oxygen of the guanosine nucleophile with a sulfur atom reduced the rate of the reverse reaction in the presence of the hard acid, namely, Mg2+ ions, and an efficient cleavage was restored by Mn2+ ions (66). This result suggests that a Mg2+ ion at site (b) coordinates with the 3'-oxygen of the guanosine nucleophile to activate the first step. Next, the 3'-bridging phosphorothiolate substrate (3'-S substrate), in which the 3' leaving oxygen had been replaced by a sulfur atom, had a dramatically reduced cleavage rate for the forward reaction in the presence of Mg2+ ions. An efficient cleavage was restored by Mn2+ ions (63,75). This result suggests that a Mg2+ ion at site (a) in Figure 4A(ii) coordinates with the 3'-leaving oxygen during cleavage. These observations can be explained by the double-metal-ion model, in which one Mg2+ ion coordinates with the nucleophile to activate the attacking group and the other Mg2+ ion coordinates with the 3'-leaving oxygen to stabilize the developing negative charge during RNA cleavage.
The possibility of coordination of a Mg2+ ion at site (d) in Figure 4A(ii) with the pro-Sp oxygen was suggested on the basis of the following experimental data. The RpS substrate, in which the pro-Rp oxygen at the scissile phosphate had been replaced by sulfur, was cleaved at a modestly reduced rate (76). By contrast, the SpS substrate, in which the pro-Sp oxygen at the scissile phosphate had been replaced by sulfur, had a drastically reduced cleavage rate in the presence of Mg2+ ions (71,75,77). Furthermore, the SpS/3'-S substrate, in which not only the pro-Sp oxygen but also the 3'-leaving oxygen had been replaced by sulfur atoms at the same scissile phosphate, was cleaved at a lower rate than the 3'-S substrate in the presence of Mn2+ ions on a background of Mg2+ ions. An efficient cleavage of the SpS/3'-S substrate, with the double-thio substitution, was restored by Zn2+ or Cd2+ ions, which are more thiophilic than Mn2+ ions, on a background of Mg2+ ions (75). Thus, a thio effect seemed apparent at the pro-Sp oxygen, and rescue both by Cd2+ and by Zn2+ ions was also evident. These results suggested that a Mg2+ ion(s) might coordinate with the pro-Sp oxygen, as well as with the 3'-leaving oxygen.
However, in consideration of the above results, we should note that an efficient cleavage of the SpS substrate, with the single-thio substitution, could not be restored either by Mn2+ ions or by Zn2+ or Cd2+ ions (75). This observation prevents us from ruling out the possibility that a Mg2+ ion does not coordinate with the pro-Sp oxygen in a direct manner during the first step in cleavage by the group I intron ribozyme (72). Further investigations are needed to determine whether direct coordination of a Mg2+ ion occurs at site (d).
Additional coordination has also been proposed at the catalytic site. A Mg2+ ion at site (c) in Figure 4A(ii) might interact directly with the 2'-OH of the guanosine, as suggested by experiments with a 2'-amino-2'-deoxyguanosine substrate and various metal ions in the ribozyme reaction (78–80). The cleavage rate was reduced by replacement of the 2'-OH with 2'-NH2 on a background of Mg2+ ions. An efficient cleavage was restored by addition of soft Mn2+ or Zn2+ ions (78,79). This result suggests that a metal ion at site (c) coordinates directly with the 2'-OH.
In addition to the coordination of metal ions discussed above, other interesting interactions have been proposed. Linear free-energy analysis of the cleavage of oligonucleotide substrates with a series of 2'-substituents at U–1 indicated that the effect on the rate of the 2'-OH group is larger than might be expected from simple inductive effects (81). The weaker electron-withdrawing 2'-OH enhanced the chemical cleavage step to a greater extent than did the more strongly electron-withdrawing 2'-F atom of the corresponding 2'-deoxy-2'-fluoro derivative. Therefore, the possibility was recently examined of a symmetrical transition state, in which the 2'-OH of U–1 might or might not interact with a metal ion [as observed at site (c) in Fig. 4A(ii)] (71). Despite the absence of lone-pair electrons at the 2'-NH3+ group that need to interact with a metal ion, the higher reactivity of the substrate with a 2'-deoxy-2'-NH3+ group than that of the substrate with a 2'-OH group at U–1 suggested that interaction of a metal ion with the 2'-OH of U–1 might not be important for catalysis by the group I intron ribozyme. The higher reactivity of the 2'-NH3+ derivative suggests that donation of a hydrogen bond from the 2'-group to the neighboring 3'-leaving oxygen might allow specific stabilization of the transition state relative to the ground state, thereby facilitating the chemical cleavage step.
The 2'-OH of U–1, the 2'-OH of A207 and the exocyclic amino group of G22 have been referred to as a catalytic triad (70). However, the observation that the chemical cleavage step with a 2'-NH3+ derivative is faster than that with the substrate with a 2'-OH (the natural substrate), despite the absence of lone-pair electrons at the 2'-NH3+ group that can accept a hydrogen bond from A207-OH, suggests another possibility for the arrangement of active-site groups within this network of interactions (71,72).
Even though the ribozyme-mediated chemical cleavage step with the 2'-OH group at U–1 (the natural substrate) is significantly (1000-fold) faster than that with 2'-H, with the metal-binding site (a) in Figure 4A(ii) being occupied by a Mn2+ ion, the rate constants for reactions with the 3'-S substrates are similar, irrespective of whether there is a 2'-OH or 2'-H at U–1. Moreover, in the presence of Mg2+ ions, with the metal-binding site (a) being unoccupied by a metal ion, the rate constants for reactions with the 3'-S substrates are similar with a 2'-OH or with a 2'-H at U–1, indicating that the 2'-OH at U–1 does not contribute significantly to the chemical cleavage of the phosphorus–sulfur bond with the 3'-mercapto leaving group. Since sulfur is a weaker acceptor of a hydrogen bond than is oxygen and, furthermore, since sulfur is a significantly better leaving group than oxygen, the 3'-mercapto leaving group suppresses the catalytic advantage provided by a hydrogen bond from the 2'-OH in the native transition state (71).
The second step of the splicing reaction is catalyzed within the same catalytic site as the first step (18,82,83). Moreover, in the presence of Mg2+ ions, both the reverse reaction of the first step and the forward reaction of the second step were inhibited with the RpS substrate (note that the pro-Rp oxygen at these steps corresponds to the pro-Sp oxygen in the forward reaction of the first step). These observations indicate that the stereochemical requirements are the same in both reactions (60,63,76,84). Therefore, the mechanism of the second step is considered to be analogous to the mechanism of the first step (18).
It is clear that the analysis of thio effects, rescue experiments and other experiments with derivatives have contributed significantly to our understanding of the mechanism of action of the large group I intron ribozyme of Tetrahymena. All the available data appear to support the refined double-metal-ion mechanism of catalysis (18) that is shown in Figure 4A(ii).
THE GROUP II INTRON RIBOZYME
In the splicing reactions catalyzed by the group II intron ribozyme (Fig. 4B), the first step was blocked when the RpS substrate was used (61) and a small thio effect was observed with the SpS substrate (64), suggesting that the pro-Rp oxygen makes an essential interaction in the transition state and that the pro-Sp oxygen is also involved in some kind of interaction. No cleavage of the 3'-S substrate was observed in the presence of Mg2+ ions but the reaction was rescued by Mn2+, Zn2+ or Cd2+ ions (67). This result indicates that a Mg2+ ion acted to stabilize the 3'-leaving oxygen by direct coordination.
The second step was monitored by a tripartite assay (trans-splicing), in which an oligonucleotide that corresponded to the 3'-splice site was added after the formation of the ribozyme/5'-exon RNA complex, because the second step might be masked by the rate-limiting conformational rearrangement between the first step and the second step that was observed in the bimolecular assay (cis-splicing). In contrast to the results of analysis of the first step, the cleavage of the RpS substrate was strongly inhibited but the SpS substrate had essentially no inhibitory effect (85). The actual stereospecificity for the thio substitution is reversed between the first step and the second step (both steps were inhibited in the reaction with the RpS substrate; note that the pro-Rp oxygen in the forward reaction of the first step corresponds to the pro-Sp oxygen in the reverse reaction of the first step and in the forward reaction of the second step). Thus, the second step is not the reversal of the first step, unlike results for the group I intron ribozyme. Even though the pro-Rp oxygen atom appears to make an essential interaction in the transition state, the nature of this interaction has not been defined since no rescue by thiophilic metal ions can be observed.
The group II intron ribozyme can also hydrolyze the bond between spliced exons [the spliced-exon reopening (SER) reaction, which corresponds to the reverse reaction of the second step]. This SER reaction proceeds with the RpS substrate but not with the SpS substrate (64). Since this step would be expected to be blocked with the RpS substrate if it followed the same reaction pathway as that of the first step of the splicing reaction, the observed SER reaction supports the conclusion that the second step is not the reversal of the first step, as mentioned above.
The substitution of the 2'-OH of the leaving ribonucleoside (the U residue of the intron in Fig. 4B) at the 3'-splice site with a hydrogen atom reduced the rate of the second step 
700-fold (85). Moreover, even though substitution with a methoxy group or a fluorine atom, respectively, reduced the rate similarly to or significantly more than substitution with a hydrogen atom, substitution with an amino group resulted in a rate that was 10-fold higher than that with a hydrogen atom (85). These results suggest that the ability to donate a hydrogen bond from the 2'-OH group is important. The 2'-amino substrate was cleaved faster in the presence of Mn2+ ions than in the presence of Mg2+ ions at higher pH (at higher pH, the 2'-amino group exists in a neutral form, as -NH2, and not in the protonated form, -NH3+) since a Mn2+ ion binds to the 2'-NH2 group better than a Mg2+ ion (nitrogen is a softer base than oxygen and the Mn2+ ion is a softer acid than the Mg2+ ion). This interaction with the 2'-oxygen at the second step involves a single metal ion in the transition state, as indicated by the fact that the dependence on the concentration of Mn2+ ions with the 2'-amino substrate on a background of Mg2+ ions is consistent with a single-metal-ion exchange (85).
The 3'-S substrate reduced the cleavage rate of the second step by 100-fold in the presence of Mg2+ ions, and the reaction was rescued completely by the addition of Mn2+, Co2+ or Cd2+ ions in the tripartite assay (67,85). However, this substrate had no effect on the bimolecular cis-splicing assay in which the rate-limiting step appeared to be the conformational rearrangement (67). This result indicates that Mg2+ ions also acted in the second step to stabilize the 3'-leaving oxygen by direct coordination, as was the case in the first step. This interaction with the 3'-oxygen also involved a single Mn2+ ion in the transition state, as observed for the cleavage of the 2'-amino substrate (85). We should emphasize, however, that the preferences of the 3'-S substrate for metal ions differed between the first step (Mn2+, Zn2+ or Cd2+) and the second step (Mn2+, Co2+ or Cd2+), indicating the differences between the environments of metal ions at the two different transition states for each splicing step.
The moiety that activates the attacking nucleophile in the two independent splice steps of the reaction of the group II intron ribozyme remains to be identified but it is apparent that a Mg2+ ion binds to the leaving 3'-oxygen to stabilize the transition state at each step.
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THE RNASE P RIBOZYME |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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In reactions catalyzed by the RNA subunit of bacterial RNase P, there is a requirement for both divalent cations (e.g., Mg2+ or Mn2+) and monovalent cations (e.g., K+ or NH4+) (62). Monovalent cations appear to be involved in the stabilization of the structure during the cleavage reaction in the absence of proteins in vitro. By contrast, divalent metal cations are required for the chemical cleavage itself, and not only for structural stabilization. During the chemical cleavage, a hydroxide ion, activated by metal ions, is thought to act as a nucleophile (86,87). Though the details of the reaction mechanism are not fully understood, it has been proposed that three Mg2+ ions participate in the transition state, because the slope of the Hill plot for the cleavage rate versus the concentration of Mg2+ ions was 3.2, and that one of the catalytic Mg2+ ions coordinates directly to the pro-Sp oxygen at the scissile phosphate. 2'-Deoxy substitution at the cleavage site reduced the apparent number of bound Mg2+ ions and decreased the apparent affinity for Mg2+ ions, suggesting that the 2'-oxygen might be one of the Mg2+-binding sites. Furthermore, 2'-methoxy substitution at the cleavage site decreased the cleavage rate, suggesting that the 2'-OH might be involved in stabilizing the 3'-leaving oxygen as the donor of a hydrogen bond (87).
According to a recent report, the cleavage rate of the RpS substrate in the presence of Mg2+ ions was at least 1000-fold lower than the cleavage rate of the natural substrate. The reduction was, however, rescued by thiophilic metal ions, such as Cd2+ and Mn2+ ions (background Mg2+ ions are needed for rescue in the case of RNase P ribozyme from Bacillus subtilis), suggesting the direct coordination of a metal ion to the pro-Rp oxygen (88,89). Since the Hill coefficient for Cd2+ rescue was 1.8, it was proposed that two metal ions coordinate to the pro-Rp oxygen in a modified model, which is consistent with the two-metal-ion model of Steitz and Steitz (18). By contrast, the cleavage reaction was also blocked with the SpS substrate (with reduction of the binding affinity of the substrate for the ribozyme in the ground state in the case of the RNase P ribozyme from Escherichia coli) and the cleavage site was shifted in the 5’ direction. The reduced rate of cleavage of the SpS substrate was not enhanced by Cd2+ and Mn2+ ions, an observation that suggests the possibility of a crucial role for the pro-Sp oxygen in stabilization of the transition state or that might be attributable to the steric exclusion of catalytic metal ions (88,89). Similar effects of RpS and SpS substrates were also observed with the eukaryotic nuclear RNase P ribozyme, in which the RNA is thought to be the catalytic component and to be evolutionarily related to the bacterial RNase P ribozyme (90). However, no thio effect was observed in the case of RNase P from plant chloroplasts, whose catalytic component appears to be a protein (91).
The 3'-S substrate also prevented cleavage at the correct site by the bacterial RNase P ribozyme and the cleavage site was moved to the next unmodified phosphodiester bond in the 5'-direction completely. The reduction in the cleavage rate was not rescued by thiophilic Cd2+ or Mn2+ ions (92). While the absence of rescue by thiophilic metal ions does not reveal the molecular nature of the inhibitory effects (the thio effect and shifting of the cleavage site), prevention of binding of a Mg2+ ion to the 3'-leaving atom as a result of the thio substitution provides one possible explanation. In addition, it is possible that several chemical and structural changes occur upon the introduction of a bulky sulfur atom.
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SMALL RIBOZYMES |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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Hammerhead, HDV, hairpin and VS ribozymes are categorized as small ribozymes because they are smaller than the ribozymes discussed above. Each of these naturally existing ribozymes catalyzes the endonucleotic cleavage of RNA via a mechanism that involves nucleophilic attack by a 2'-OH group on the phosphorus of the neighboring phosphodiester bond, generating 5'-OH and 2',3'-cyclic phosphate termini (for reviews, see 4,25,93). The cleavage reactions catalyzed by these ribozymes appear to proceed with inversion of the configuration at the phosphorus atom suggesting a direct in-line attack with development of a pentacoordinate transition state or intermediate (42,94–97).
The smallest of the naturally occurring catalytic RNAs that have been identified to date are the hammerhead ribozymes (Fig. 1A), which were found in several plant viral satellite RNAs, a viroid RNA and the transcript of a nuclear satellite DNA of a newt (98; for reviews, see 3,4). These ribozymes have been extensively investigated, in particular with respect to the mechanism of action of catalytic metal ions (17,25,27,45,46).
Hammerhead ribozymes have a basic requirement for divalent metal ions, such as Mg2+ ions (5,6,17–22,24,25,27,45,99–104). In studies of the hammerhead reaction, the relationship at a certain pH between the 
pKa values of metal ions in water and the difference in the observed rates of cleavage in the presence of the corresponding metal ions suggested a single-metal-ion mechanism in which Mg2+-hydroxide acts as a general base catalyst (17). However, it was also noted that a general double-metal-ion mechanism, in which metal ions act as Lewis acids that coordinate directly to the 2'-OH and the 5'-leaving oxygen, for activation of a nucleophile and for stabilization of a developing negative charge on the leaving group, respectively, might also explain reactions catalyzed by hammerhead ribozymes (20–22,24,25).
It should also be noted that, under extreme conditions (in the presence of 1–4 M monovalent cations, such as Li+, Na+ and NH4+), hammerhead ribozymes do not require divalent metal ions for catalysis (43). On the basis of this observation, some researchers have claimed that hammerhead ribozymes are not metalloenzymes (see below).
HDV ribozymes are derived from the genomic and the antigenomic RNAs of hepatitis delta virus (105–108). In studies of reactions catalyzed by HDV ribozymes, three groups demonstrated recently that an intramolecular functional group, namely N3 at C76 in the antigenomic HDV ribozyme and N3 at C75 in the genomic HDV ribozyme, can, in fact, act as a true catalyst (40–42). However, with respect to the roles of these N3s, two different mechanisms, namely general base catalysis and general acid catalysis, were proposed. In the former scenario, it was proposed that the deprotonated N3 of C76 might be involved in cleavage as a general base that abstracts a proton from the 2'-OH to promote its nucleophilic attack on the scissile phosphate in the transition state of reactions catalyzed by the antigenomic HDV ribozyme (41). In the latter case, it was proposed that the protonated N3 of C75 in the genomic HDV ribozyme might act as a general acid to stabilize the developing negative charge at the 5'-leaving oxygen and that a metal ion might act as a general base (42). However, although further investigations are required, there remains the possibility that the catalytic mechanism of the antigenomic ribozyme is the same as that of the genomic ribozyme (109).
In discussion of the reaction catalyzed by the genomic HDV ribozyme, the importance has been emphasized of the neutralization of the substantial negative charge that develops on the 5'-leaving oxygen and the essential role of general acid catalysis in the cleavage of RNA (42). Such issues should be relevant not only in the case of reactions catalyzed by HDV ribozymes but also in the case of reactions catalyzed by other small ribozymes since cleavage of the bond between phosphorus and the 5'-oxygen is the overall rate-limiting step, as shown in Figure 2 (see below; 22,25,56). The efficient cleavage of a phosphodiester bond requires both the activation of the 2'-attacking oxygen and the stabilization of the 5'-leaving oxygen.
Hairpin ribozymes were originally derived from the minus strand of the satellite RNA of tobacco ringspot virus (sTRSV), chicory yellow mottle virus type 1 (sCYMV1) and arabis mosaic virus (sArMV) (110–113). Hairpin and hammerhead ribozymes can also catalyze the ligation of cleaved products, with the ligation efficiency being much higher for the hairpin ribozyme than the hammerhead. The ligation reaction is thought to be the reverse of the cleavage reaction since it uses the same termini as those produced upon cleavage. Hairpin ribozymes favor the ligation reaction rather than cleavage (ligation occurs 10-fold faster than cleavage). By contrast, hammerhead ribozymes favor the cleavage reaction [cleavage occurs 
100-fold faster than ligation (47,114–116)]. The ratio of equilibrium constants (kcleavage/kligation) can be explained by the differences between entropies: the loss of entropy that occurs with ligation is smaller for the hairpin than for the hammerhead ribozyme, indicating that the more rigid hairpin structure undergoes a smaller change in dynamics on ligation than the more flexible hammerhead (117). Catalysis by hairpin ribozymes in the absence of metal ions has been reported by several groups independently (34–39). Hairpin ribozymes can be considered to be a distinct class of ribozymes that do not require metal ions as cofactors (118). The catalyst(s) seems to be a nucleobase(s).
The VS ribozyme originated from the mitochondria of certain isolates of Neurospora (119). The reaction catalyzed by the VS ribozyme requires a divalent cation such as the Mg2+ ion (120). Some regions that are important for catalysis and some interactions between the phosphate backbone and metal ions have been identified (121–123). The reaction appears to be independent of pH but the possibility exists for a conformational change prior to cleavage that might mask a dependence on pH (120,124). The catalytic group(s) in the cleavage reaction has not yet been unambiguously identified.
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REACTIONS CATALYZED BY HAMMERHEAD RIBOZYMES |
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TOPABSTRACTINTRODUCTIONCLEAVAGE OF THE PHOSPHODIESTER...POSSIBLE CATALYTIC FUNCTIONS OF...LARGE RIBOZYMESTHE MECHANISM OF...THE RNASE P RIBOZYMESMALL RIBOZYMESREACTIONS CATALYZED BY...HDV RIBOZYME-CATALYZED REACTIONSTHE HAIRPIN RIBOZYMEREACTIONS CATALYZED BY RIBOZYMES...CONCLUSIONSREFERENCES |
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Hammerhead ribozymes are among the smallest catalytic RNAs. The sequence motif, with three duplex stems and a conserved core of two non-helical segments that are responsible for the self-cleavage reaction (cis-action), was first recognized in the satellite RNAs of certain viruses (3). Engineered trans-acting hammerhead ribozymes, consisting of antisense sections (stem I and stem III) and a catalytic core with a flanking stem–loop II section (Fig. 1A, left), have been used in mechanistic studies and tested as potential therapeutic agents (13,46). Hammerhead ribozymes cleave their target RNAs at specific sites, generating a 2',3'-cyclic phosphate and a 5'-OH terminus. The NUH rule, where N can be any nucleotide and H can be A, U or C, was originally proposed to define sites of cleavage, with the most efficient cleavage occurring at GUC triplets (125–130). However, the NUH rule was reformulated into the NHH rule since other triplets, such as GAC and GCC, can also be cleaved by a hammerhead ribozyme (131).
Over the past few years, several attempts have been made to determine the overall global structure of hammerhead ribozymes (99,132–137). Although initial structural studies indicated that possible configurations of the scissile phosphate did not allow for an in-line attack mechanism, recent crystallographic studies of a ribozyme with a product or with a modification (known as a kinetic bottleneck modification) adjacent to the cleavage site succeeded in trapping an intermediate that more closely resembled the transition state (137; reviewed in 138). However, even in this case, the intermediate cannot be considered as a real transition-state intermediate. In all crystals of ribozymes examined to date, a 
-shaped configuration has been identified, with stem I forming an acute angle with stem II, and stems II and III being stacked coaxially to form a pseudo-A-form helix, in agreement with results inferred from studies of fluorescence energy transfer and from electrophoretic and chemical cross-linking studies (Fig. 1A, right) (26,139–142). Such studies indicate the involvement of two reversed-Hoogsteen G·A base pairs between G8·A13 and A9·G12, as well as a non-Watson–Crick A14·U7 base pair that is formed by a single hydrogen bond. These base pairs are followed by stem II and are stacked ‘coaxially’ onto the non-Watson–Crick A15.1·U16.1 base pair, with resultant formation of a pseudo-A-form helix by stems II and III. Four sequential nucleotides (C3U4G5A6) form a ‘uridine-turn’ motif, allowing the phosphate backbone to turn and connect with stem I. The uridine-turn forms a catalytic pocket into which the nucleobase at the cleavage site, namely C17, is inserted (133). The crystal structure of the enzyme–product complex of the hammerhead ribozyme has been determined (137). The structure suggests that the distance between C17 and G5/A6 in the transition state is smaller than previously proposed and that dramatic conformational changes, which include C17, occur in the pathway from the ground state to the transition state.
Structural metal ions in reactions catalyzed by hammerhead ribozymes
It is generally accepted that the tertiary structures of RNA molecules are stabilized by metal ions. The roles of metal ions in ribozyme-catalyzed reactions are of two distinct types: metal ions can act as catalysts during the chemical cleavage step, as shown in Figure 3; and they can also stabilize the conformation of the ribozyme–substrate complex. The ion-dependent changes in the conformation of a hammerhead ribozyme can be easily followed by monitoring the influence of metal ions on its electrophoretic mobility (26). The effects of metal ions on the formation, upon subsequent addition of Mg2+ ions, of an active complex between a hammerhead ribozyme and its substrate can also be monitored by NMR spectroscopy (143–152).
Binding sites for metal ions have been identified by capturing metal ions within the crystal structure, and such capture provides an indication of the importance of the metal ions in catalysis (99,132–136). It was proposed that a Mg2+ ion binds to the pro-Rp oxygen of the 5'-phosphate of A9 (P9 phosphate) with further hydrogen bonding associated with N7 of G10.1 (153–155). Another site for a Mg2+ ion was localized in the vicinity of the cleavage site. It was proposed that, at this cleavage site, a Mg2+ ion binds directly to the pro-Rp oxygen of the scissile phosphate. Although this possibility remains to be confirmed, the function of this second metal ion near the scissile phosphate has been proposed to be activation of the attacking 2'-OH in the transition state. The site of yet another metal ion has also been proposed. Such an ion would act as a switch that induces the conformational changes required to achieve the transition state; it would be located adjacent to G5 in the catalytic core. This last putative site was identified from an analysis of the kinetics of a Tb3+ inhibition experiment and the elucidation of the crystallographic structure of the complex (136). The coordination of a metal ion at this site in solution was also investigated by lanthanide luminescence spectroscopy (156). An additional metal ion binding site in the hammerhead ribozyme has also been identified by 31P NMR spectroscopy (149). In this case, the metal ion is associated with the A13 phosphate in the catalytic core with an apparent Kd of 250–570 µM. However, the exact role of this metal ion remains unclear. It seems likely that it might be involved in structural folding since a structural change was detected at this site upon the binding of the metal ion.
The importance of the binding of a metal ion at the P9 phosphate, not only in the ground state but also in the transition state, was demonstrated by kinetic analysis with a modified hammerhead ribozyme with a phosphorothioate modification at this site (157,158) and, in parallel, by analysis with a ribozyme with an abasic mutation at this site (159,160). The binding of a metal ion (Cd2+) to the Rp sulfur of the P9 phosphorothioate in the transition state was much stronger than the binding in the ground state, suggesting the existence of additional ligands for the metal ion in the transition state. Moreover, our own studies indicate strongly that the binding of a metal ion to N7 of G10.1 is catalytically important but not indispensable: cleavage still occurred with a minimally modified ribozyme, in which N7 of G10.1 was merely replaced by C7 (introduction of an N7-deazaguanine residue to prevent the metal ion from binding to this site) (161). Cleavage was retarded, with 30-fold reduction in the rate of cleavage by the modified ribozyme. By contrast, a 1000-fold reduction in the cleavage rate resulted from the introduction of an Rp-phosphorothioate at the P9 site (157).
It was proposed very recently that the first metal ion that binds to the P9 phosphate in the ground state shifts toward a non-bridging pro-Rp oxygen at the scissile phosphate during the reaction and binds to this pro-Rp oxygen in the transition state. This scenario is consistent with the prediction that additional binding to this P9 metal ion must occur in the transition state, as mentioned above. Furthermore, it was also proposed that the metal ion at P9 must be involved directly in the chemical cleavage step, acting as a base catalyst in the transition state (27), despite the fact that the P9 metal ion is located at a distance of 20 Å from the scissile phosphate in the ground state. However, these proposals have been questioned by other investigators (162,163).
Although it was predicted that a hydrated Mg2+ ion should participate directly in catalysis, acting as a base catalyst in deprotonation of the 2'-OH of C17, no crystal structure has been obtained with a trapped metal ion located at this cleavage position that might confirm the direct involvement of such a metal ion in deprotonation of the 2'-OH (137).
The rate-limiting departure of the 5'-oxygen in reactions catalyzed by hammerhead ribozymes
In the non-enzymatic hydrolysis of a natural RNA, the cleavage of the P-O(5') bond is the overall rate-limiting step in hydrolysis. By contrast, attack by the 2'-OH on the phosphorus atom is the rate-limiting step with a 5'-S substrate that contains a phosphorothiolate substitution (54–56,164,165) since, as mentioned above, the latter is hydrolyzed much more rapidly than the natural substrate (Fig. 2). The same technique as that used to draw these conclusions was used to determine the rate-limiting step in reactions catalyzed by hammerhead ribozymes.
It was reported that, in the hammerhead-catalyzed cleavage reaction, the departure of the 5'-leaving group is not rate-limiting and that a metal cofactor does not interact with the leaving group (45,100,101). This conclusion was based on experiments with the 5'-S almost-DNA substrate, that was an oligodeoxynucleotide substrate that contained a 5'-bridging phosphorothiolate linkage adjacent to one ribonucleotide at the cleavage site (45). No appreciable thio effect was observed and no preference was detected for either Mg2+ or Mn2+ ions (100,101). However, the 5'-S almost-DNA substrate basically consisted of DNA and the ribozyme reaction with this substrate had an unusually limited dependence on pH. Thus, we might expect that observed rates of reaction might reflect steps other than the chemical cleavage step. In the case of a 5'-S RNA substrate consisting only of RNA, the rate of ribozyme-mediated cleavage of the 5'-S RNA substrate in the presence of Mg2+ ions was higher by almost two orders of magnitude than that of cleavage of the natural substrate (56). If TS1 were a higher energy state than TS2 in the ribozyme reaction with the natural substrate, the cleavage rate for the 5'-S RNA substrate should be similar to that for the natural substrate because the 5'-bridging phosphorothioate linkage would not be expected to enhance the attack by the 2'-OH (53). By contrast, if TS2 were a higher energy state than TS1 in the ribozyme reaction with the natural substrate, we would expect that the rate of cleavage of the 5'-S RNA substrate would be much higher than that of the natural RNA substrate because a mercapto group is a better leaving group than a hydroxyl group. On the basis of these considerations, the results indicate that TS2 is a higher energy state than TS1 in the reactions of hammerhead ribozymes with natural substrates, as indicated in Figure 2.
Catalytic metal ions in reactions catalyzed by hammerhead ribozymes: single-metal-ion mechanisms
In the case of the proteinaceous enzyme RNase A, which does not require metal ions as cofactors, the acid/base catalysts are provided by two histidine residues within the catalytic pocket (Fig. 5A). Such acid/base functionality can, in principle, be replaced by Mg2+-bound water molecules. The generally accepted mechanism of hammerhead ribozyme reactions is a single-metal-ion mechanism (27,45,99–103). In this proposed mechanism, the hydroxide ion of a hydrated Mg2+ ion acts as a general base to deprotonate the attacking 2'-OH; the Mg2+ ion coordinates directly to the pro-Rp oxygen at the scissile phosphate, acting as an electrophilic catalyst in TS1; and it is not a metal ion but a proton that acts as a general acid to stabilize TS2 (Fig. 5B).
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The single-metal-ion mechanism is supported by the fact that no metal ion was found close to the 5'-leaving oxygen in the original crystallographic structure of a freeze-trapped conformational intermediate of a hammerhead ribozyme (99). Based upon the crystal structure and a molecular dynamic simulation, a different single-metal-ion mechanism was proposed as follows (102,103). It was suggested that the involvement of just one metal ion in the transition state might be sufficient for cleavage. In this model, one Mg2+ ion coordinates simultaneously and directly to the pro-Rp oxygen and to the 2'-attacking oxygen at the cleavage site, acting as a Lewis acid to enhance the deprotonation of the 2'-OH and the subsequent attack by the nucleophile on the phosphorus atom and/or to stabilize the transition state. In addition, it was also proposed that one of the outer-sphere water molecules that surrounds the metal ion might be located at a position such that it can act as a general acid to donate a proton to the 5'-leaving group (102,103)
Another model for cleavage by hammerhead ribozymes was proposed that was based on the results of molecular dynamic studies (166). This model involves two metal ions but it is reminiscent of the single-metal-ion mechanism. In this model, two metal ions are bridged by a hydroxide ion (the µ-hydroxo-bridged Mg2+ cluster). One of the metal ions, located near the scissile phosphate, binds to the pro-Rp oxygen to act as an electrophilic catalyst. The bridging OH– between the two Mg2+ ions abstracts the proton from the Mg2+-bound water molecule. Then the activated hydroxide ion associated with the Mg2+ ion deprotonates the proximal 2'-OH at the cleavage site to activate the nucleophile, acting as a base catalyst.
As described in the previous section, formation of TS2 is the rate-limiting step in non-enzymatic reactions (25,54–56). Thus, TS2 must somehow be stabilized energetically for effective catalysis. Therefore, the hypothesis that reactions catalyzed by hammerhead ribozymes involve only a general base is insufficient.
Catalytic metal ions in reactions catalyzed by hammerhead ribozymes: double-metal-ion mechanisms
Double-metal-ion mechanisms, in which two metal ions are involved in the chemical cleavage step, have been proposed by numerous groups of investigators (5,18–22,24,25,104). From molecular orbital calculations and kinetics analysis, our group postulated that the direct coordination of Mg2+ ions with the attacking or the leaving oxygen might promote formation or cleavage of the P-O bond, with these ions acting as Lewis acids (Fig. 5C) (5,20,22,25,167). Moreover, we excluded the possible coordination of metal ions, as electrophilic catalysts, to the pro-Rp oxygen at the scissile phosphate bond (5,25,162,168,169). Studies of solvent isotope effects and kinetic analysis of a modified substrate (phosphorothiolate; 5'-S substrate), with a 5'-mercapto leaving group at the cleavage site, provided strong support for the double-metal-ion mechanism of catalysis (20,22,25).
The overall transition state structure in the hammerhead cleavage reaction is TS2, regardless of whether the reaction proceeds via a concerted one-step mechanism or via a two-step mechanism with a stable pentacoordinated intermediate. Our analysis with a 5'-S substrate demonstrated that it is important that any enzyme that catalyzes the hydrolysis of RNA should stabilize TS2 by donating a proton to the 5'-leaving oxygen or by ensuring coordination of a metal ion to the leaving oxygen. This acid catalysis by a metal ion is also supported by a recently determined crystallographic structure, in which a Co2+ ion was located close to the 5'-leaving-oxygen atom of the scissile phosphate (134).
Figure 6A shows experimentally derived profiles of pH versus rate for reactions in H2O and D2O (20,25,169). The magnitude of the apparent isotope effect (ratio of rate constants in H2O and D2O) is 4.4 and the profiles appear to support the possibility that a proton is transferred from (Mg2+-bound) water molecules. However, careful analysis led us to conclude that a metal ion binds directly to the 5'-oxygen. Since the concentration of the deprotonated 2'-oxygen in H2O should be higher than that in D2O at a fixed pH, we must take into account this difference in pKa, namely, pKa (pKaD2O – pKaH2O), when we analyze the solvent isotope effect of D2O (20,25,169,170). We can estimate the pKa in D2O from the pKa in H2O using the linear relationship shown in Figure 6B (6,25,169–172). If the pKa for a Mg2+-bound water molecule in H2O is 11.4, the pKa is calculated to be 0.65 (Fig. 6B, red line). Then, the pKa in D2O should be 12.0. Demonstrating the absence of an intrinsic isotope effect (kH2O/kD2O = 1), the resultant theoretical curves closely fit the experimental data, with an apparently 4-fold difference in observed rate constants (Fig. 6A). This result indicates that no proton transfer occurs in the hammerhead ribozyme-catalyzed reaction in the transition state and supports the hypothesis that the metal ions function as Lewis acids. In Figure 6A, the apparent plateau of rate constants above pH 8 reflects the disruption of the active hammerhead complex by the deprotonation of uridine and guanosine residues.
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= kH2O/kD2O = 1; where The double-metal-ion mechanism is also supported by results reported by Pontius et al. (21) and Lott et al. (24). They pointed out the minimal likelihood of base catalysis by Mg2+-bound hydroxide that deprotonates the attacking 2'-OH and the strong likelihood of Lewis acid catalysis by the direct coordination of a Mg2+ ion with the attacking 2'-OH, which enhances the deprotonation of the nucleophilic 2'-OH. Their suggestions were based on the inverse correlation between the rate of cleavage and the pKa of the added metal ions. Their argument was as follows. One of the observations used to support the single-metal-ion mechanism of catalysis (Fig. 5B) is that the lower the pKa, the higher is the cleavage rate at a given concentration of metal ions. Although metal ions with lower pKa values might be present at higher concentrations in the form of solvated metal hydroxides at a given pH, such ions should be correspondingly weaker bases and, therefore, they should be less able to remove the proton from the 2'-OH. As a result, the effect of concentration would be reduced by the effect of basicity. In other words, the dependence on pKa cannot be adequately explained by the hypothesis that the solvated metal hydroxide acts as a base in catalysis. By contrast, deprotonation of the 2'-OH can be greatly accelerated by its direct binding to a metal ion, in particular, a metal ion with a relatively low pKa, because the pKa of a 2'-OH with a bound metal ion can be reduced by several units. Such arguments are further supported b