Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Abstract

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this study, we have examined the role of cytosine in more detail with the antigenomic ribozyme. Evidence that proton transfer in the rate-determining step involved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76 Delta) ribozymes in D(2)O and H(2)O. In both reactions, a similar kinetic isotope effect and shift in the apparent pKa indicate that the buffer is functionally substituting for the side chain in proton transfer. Proton inventory of the wild-type reaction supported a mechanism of a single proton transfer at the transition state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. For the imidazole analogues that rescued activity, the apparent pKa of the rescue reaction, measured under k(cat)/K(M) conditions, correlated with the pKa of the base. From these data a Brønsted coefficient (beta) of 0.51 was determined for the base-rescued reaction of C76 Delta. This value is consistent with that expected for proton transfer in the transition state. Together, these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wild-type ribozyme to facilitate RNA cleavage.

Figure 1

Sequence and secondary structure of the HDV antigenomic ribozyme PEX1 with C76 emphasized. Numbering is from the cleavage site (indicated by an arrow) such that cleavage occurs between positions −1 and 1.

Figure 2

Kinetic solvent isotope effect for wild-type and imidazole-rescue reactions of C76Δ. (A) Second-order plot of C76Δ cleavage by imidazole free base in H₂O. The apparent second-order rate constant () was plotted against fraction of free base. For wild type (B) and C76Δ (C), first-order (k_obs) and apparent second-order () rate constants in H₂O (○) and D₂O (□) were plotted against pL (L = H, D). A ΔpKa ≈ 0.6 was consistent with the expected value of 0.6 for an amino group. (D) Proton inventory of the wild-type PEX1 reaction. The first-order rate constants measured in varied solvent isotopic composition were normalized to the rate constant in H₂O () and plotted against molar ratio of D₂O (n_D2O). The data were fit to a linear dependence (n = 9; r = 0.99).

Figure 3

Brønsted analysis of C76Δ cleavage activity with imidazole analogues. (A) The apparent second-order rate constant as a function of pH. The apparent pKa of each reaction is labeled for 4-methylimidazole (squares), imidazole (circles), 4-hydroxymethylimidazole (diamonds), and 3-amino-1,2,4-triazole (triangles). (B) The Brønsted plot of C76Δ cleavage in the buffers shown in A; Brønsted coefficient β of 0.51 is determined from the slope (n = 4; r = 0.96).

Figure 4

Two kinetically equivalent mechanisms for general acid-base catalysis by RNA side chains or imidazole buffer. (A) In the C76u and C76Δ mutants, the unsaturated ring nitrogen of imidazole base accepts the proton from the nucleophilic group for general-base catalysis; in the wild type, cytosine serves a similar role. An unidentified functional group stabilizes the leaving group. (B) Imidazolium or protonated cytosine sidechain acts as a general acid by donating a proton to the leaving group. A specific base deprotonates the 2′-OH nucleophilic group at pre-equilibrium.