Use of intrinsic binding energy for catalysis by an RNA enzyme

Use of intrinsic binding energy for catalysis by an RNA enzyme

^*Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215; and ^‡Department of Biochemistry, B400 Beckman Center, Stanford University, Stanford, CA 94305-5307

Abstract

The contribution of several individual ribozyme⋅substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3′ or 5′ ends. The base pairs at positions 1.1–2.1 and 15.2–16.2, which flank the conserved core, each contribute 10⁴-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a “fraying model” in which each ribozyme⋅substrate helix can exist in either an unpaired (“open”) state or a helical (“closed”) state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of “intrinsic binding energy” from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

Footnotes

↵ † Present address: Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138.
↵ § To whom reprint requests should be addressed.
Olke C. Uhlenbeck
↵ ¶ It was previously suggested that binding of full-length oligonucleotides substrates to the hammerhead ribozyme is weaker than binding of products, consistent with a “substrate destabilization” mechanism (17). However, the support for this mechanism appears to have arisen from an experimental artifact in the measurement of dissociation rate constants (18). The binding data presented in Table 1 provide further evidence against thermodynamic destabilization that weakens binding of substrate relative to products.
↵ ‖ The rate increases observed upon adding the U of P1-GU and the C of CGUC-P2 are within a factor of 3 of that predicted from the fraying model and nearest-neighbor calculations that account for the free energy contributions from the added base pair and stacking interactions (ref. 20; calculations not shown).
ABBREVIATIONS:

Tris,

tris(hydroxymethyl)aminomethane;

Epps,

N-2-hydroxyethylpiperazine-N′-3-propanesulfonic acid;

Hepes,

N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid;

Bistris propane,

1,3-bis[tris(hydroxymethyl)methylamino]propane. P1 and P2 are the 5′- and 3′-cleavage products, respectively. The nomenclature used to describe the truncated substrates is as follows: “P1-” and “-P2” are used to represent truncated sequences that contain the entire sequence of the P1 and P2 products along with additional 3′ or 5′ residues, respectively (Fig. 1A). The additional nonconserved residues that base pair to the ribozyme to form helices I and III (Fig. 1A) and are varied in this study are underlined