A common speed limit for RNA-cleaving ribozymes and deoxyribozymes

Abstract

It is widely believed that the reason proteins dominate biological catalysis is because polypeptides have greater chemical complexity compared with nucleic acids, and thus should have greater enzymatic power. Consistent with this hypothesis is the fact that protein enzymes typically exhibit chemical rate enhancements that are far more substantial than those achieved by natural and engineered ribozymes. To investigate the true catalytic power of nucleic acids, we determined the kinetic characteristics of 14 classes of engineered ribozymes and deoxyribozymes that accelerate RNA cleavage by internal phosphoester transfer. Half approach a maximum rate constant of approximately 1 min(-1), whereas ribonuclease A catalyzes the same reaction approximately 80,000-fold faster. Additional biochemical analyses indicate that this commonly encountered ribozyme "speed limit" coincides with the theoretical maximum rate enhancement for an enzyme that uses only two specific catalytic strategies. These results indicate that ribozymes using additional catalytic strategies could be made that promote RNA cleavage with rate enhancements that equal those of proteins.

FIGURE 1.

Mechanism for RNA cleavage by internal phosphoester transfer involving the 2′-hydroxyl group. The phosphorus center of the RNA linkage (1) is attacked by the 2′-oxygen nucleophile, which generates the dianionic phosphorane species (2). This pentacoordinate structure degrades to yield 2′,3′-cyclic phosphate-terminated (3) and 5′-hydroxyl-terminated (4) RNA fragments. The four main strategies (see text) for catalytic activation of this reaction are denoted (Greek lettering and arrows) in this depiction of an otherwise uncatalyzed phosphoester transfer reaction.

FIGURE 2.

Representative examinations of the kinetic characteristics of RNA-cleaving ribozymes and deoxyribozymes. Plots 1 through 3 depict the assay data used to determine the optimal reaction conditions at pH 7.5 and 23°C with regard to the concentration (c) of enzyme, monovalent ion, and cofactor, respectively. Dashed lines identify the concentration of enzyme or cofactor needed to attain half-maximal k_obs. Solid lines reflect theoretical activity curves generated using the maximum k_obs and the K_D values derived from plots 1 and 3. Optimization reactions for HD3 progressed as follows: plot 1 (500 mM KCl, 20 mM L-histidine); plot 2 (100 nM enzyme, 20 mM L-histidine); plot 3 (100 nM enzyme, 1 M KCl). Note: the methyl ester of histidine (HME) was used in place of histidine in plot 3 to increase cofactor solubility (Roth and Breaker 1998). Optimization reactions for MR11 progressed as follows: plot 1 (250 mM KCl, 20 mM MgCl₂); plot 2 (100 nM enzyme, 20 mM MgCl₂); plot 3 (100 nM enzyme, 500 mM KCl).

FIGURE 3.

The influence of pH on the k_obs for six RNA-cleaving ribozymes and deoxyribozymes. In each panel, the sequence and secondary structure model for bimolecular constructs of the ribozyme or deoxyribozyme (bottom strand) and substrate (top strand) is depicted. An arrowhead designates the site of cleavage. The substrates for HD3 and MD1 are DNA oligonucleotides that carry RNA linkages (underlined). The plot in each panel shows the logarithm of the k_obs for enzyme catalysis as a function of pH. The dashed line identifies the pH required to attain half-maximal k_obs. Colored zones indicate the range of rate constants that are expected from enzymes that fully use the γ catalytic strategy (pink) or a combination of α and γ catalytic strategies (light blue) to the exclusion of other possible strategies. The deoxyribozyme HD3 was analyzed with HME as a cofactor; the remaining enzymes used MgCl₂. Reagent concentrations were as follows (enzyme, KCl, and cofactor, respectively): (A) HD3: 100 nM, 1 M, 400 mM; (B) MD1: 1 μM, 200 mM, 10 mM; (C) MR2: 300 nM, 150 mM, 350 mM; (D) MR4: 35 nM, 250 mM, 35 mM; (E) MR11: 100 nM, 500 mM, 750 mM; (F) C1 V2 Trans: 300 nM, 250 mM, 1 M. Below pH 7, assays of MD1 were buffered with succinate, and MR2 and MR4 with Bis-Tris.

FIGURE 4.

Putative αγ ribozymes do not exhibit metal-mediated β catalysis. (A) Schematic representation of an Sp thiophosphate RNA linkage and the possible contacts made by a metal-ion cofactor during β catalysis. The sulfur atom will be located at either of the two nonbridging positions (Rp or Sp) with near equal distribution. If the enzyme is an obligate metalloenzyme for β catalysis, then the metal ion (M) is expected to coordinate selectively with only one nonbridging position, as established by the active site. The inset reflects the fact that Mn²⁺ can interact with oxygen or sulfur with similar affinity, whereas Mg²⁺ has high affinity only for oxygen. (B–D, left) Kinetic characteristics of MR2, MR4, and MR11 ribozymes with thiophosphate substrates in the presence of Mg²⁺ (filled circles) and Mn²⁺ (open circles). The dashed line designates the plateau in RNA cleavage that would be expected if a thio effect precluded the processing of one of the two isomers. (Right) The bar graph represents the fraction of total RNA substrate processed in the presence of Mg²⁺ or Mn²⁺. The fraction cleaved was corrected and normalized as detailed in Materials and Methods. Again, the dashed line designates the maximum value for RNA cleavage that is expected if Mg²⁺ could only process one of the two thiophosphate isomers.

FIGURE 5.

Enzymes with more complicated kinetic profiles can meet or break the αγ speed limit. (A–C) Sequences, secondary-structure models, and characteristics for the MR5, 10–23, and X-motif enzymes, respectively. The cofactor and pH dependencies for each enzyme are depicted in the plots to the left, and the data resulting from thio-effect examination are given in the plots to the right. Other notations are as described in the legends to Figures 2 ▶–4 ▶. Reagent concentrations (enzyme, KCl, and cofactor, respectively) were as follows for each construct: MR5: 24 nM, 250 mM, 200 mM; 10–23: 100 nM, 0 mM, 30 mM; X-motif: 50 nM, 0 mM, 20 mM. Below pH 7, assays of MR5 were buffered with Bis-Tris. Assays of X-motif were buffered with HEPES in the range pH 6.7 to pH 8. Portions of the data presented in Figure 5C ▶ were obtained from Lazarev et al. (2003) for comparison.