Ribozyme-Mediated Cleavage of a Substrate Analogue Containing an Internucleotide-Bridging 5‘-Phosphorothioate: Evidence for the Single-Metal Model†
journal contributionposted on 23.04.1996, 00:00 by Robert G. Kuimelis, Larry W. McLaughlin
An oligonucleotide substrate containing a 5‘-bridging phosphorothioate linkage adjacent to a ribonucleotide has been used to investigate the cleavage mechanism of the hammerhead ribozyme and to probe the catalytic role of the metal cofactor(s). Specifically, we tested the hypothesis that a second metal interacts with the 5‘-leaving group to facilitate the cleavage event. To this end, we have examined the ribozyme-mediated cleavage activity of the phosphorothioate substrate at pH 7.5 with a series of divalent metals in both the presence and absence of the polycation spermine. The cleavage products are found to be the same as for the native sequence under a variety of reaction conditions. The influence of divalent metal ion concentration, temperature, and pH on the cleavage rate has also been examined for both the oxo linkage and the thio analogue. Spermine (but not spermidine or NaCl) is shown to support efficient cleavage of the thio analogue in the absence [5 mM ethylenediaminetetraacetic acid (EDTA)] of a divalent metal cofactor. The cleavage of the oxo linkage exhibits a solvent deuterium isotope effect of 3.6, but a similar effect is not observed with the thio analogue. The pseudo-first-order rate constants for cleavage of the thio analogue in the presence of 10 mM Mg2+ or Mn2+ at pH 7.5 are 65 and 82 × 10-3 min-1, respectively. The native oxo linkage is cleaved at essentially the same rate as the thio analogue (35 and 97 × 10-3 min-1 for Mg2+ and Mn2+, respectively). The absence of an appreciable thio effect and the lack of a preference for either Mg2+ or Mn2+ provides compelling evidence that the metal cofactor does not interact with the 5‘-thioanion (or oxyanion) leaving group in the transition state. These rate comparisons additionally reveal that the departure of the 5‘-leaving group is not the rate-limiting step of the cleavage reaction catalyzed by the hammerhead ribozyme.