Hammerhead Ribozyme-Mediated Cleavage of a Substrate Analog Containing an Internucleotidic Bridging 5'-Phosphorothioate: Implications for the Cleavage Mechanism and the Catalytic Role of the Metal Cofactor

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5′-oxy and 5′-thio substrates by a hammerhead ribozyme

“…3 Top Left). This conclusion was confirmed by the previous observation that 5Ј-thio substrate was more than four orders of magnitude more susceptible to nonenzymatic hydrolysis than the corresponding 5Ј-oxy substrate (23,26,27,34,35). If the formation of the pentacoordinate intermediate [P(V)] were rate-limiting (if TS1 were a higher energy state than TS2), R11S should have been hydrolyzed at a rate slower than the rate of hydrolysis of R11O because the 5Ј-bridging phosphorothioate linkage would not be expected to enhance the attack by 2Ј-oxygen (36), and because the transition state for R11S with an apical sulfur atom is expected to be less stable than the corresponding transition state for R11O with an apical oxygen (37).…”

“…The global three-dimensional structures of the two crystallized ribozymes were nearly identical: one domain of the conserved core, which consists of the sequence C 3 U and is located next to stem I, makes a sharp turn identical to the uridine turn in tRNAs (10). As a result, stem II and stem III are aligned almost colinearly through pseudocontinuous, long A-type helices.It is now well established that ribozymes are metalloenzymes (2,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Although x-ray analysis of a hammerhead ribozyme identified one potential catalytic metal ion (7-9), the exact number of metal ions required for catalysis remains obscure, because the newly captured conformational intermediate appears to demand further conformational change for the following in-line attack (9).…”

“…It is now well established that ribozymes are metalloenzymes (2,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Although x-ray analysis of a hammerhead ribozyme identified one potential catalytic metal ion (7)(8)(9), the exact number of metal ions required for catalysis remains obscure, because the newly captured conformational intermediate appears to demand further conformational change for the following in-line attack (9).…”

The hammerhead cleavage reaction in monovalent cations

“…The finding that the rate enhancement in Li ϩ approaches that in Mg 2ϩ , and that the hammerhead retains significant activity in Co(NH 3 ) 6 3ϩ , weakens the case for a metal ion acting as a base in the reaction+ Furthermore, the relationship between ionic radius and cleavage rate suggests that the differences among ions reflect their differential abilities in stabilizing the catalytically active conformation of the hammerhead+ This contrasts to the picture emerging for the hepatitis delta virus (HDV) ribozyme, a different ribozyme that also carries out site-specific cleavage leaving 59 hydroxyl and 29,39-cyclic phosphate termini (Been & Wickham, 1997)+ For the HDV ribozyme, a divalent metal ion is thought to act as a base in the cleavage reaction: removal of the divalent metal reduces the rate significantly (5,000-fold at pH 7), and exposes the underlying acid catalysis, as indicated by an inversion of the pH dependence (Nakano et al+, 2000)+ With this in mind, we investigated the pH dependence of HH16+1 in monovalent and divalent metals+ In 10 mM Mg 2ϩ , the hammerhead cleavage rate increases approximately 10-fold with each one unit increase in pH (Dahm et al+, 1993; Fig+ 2B)+ We observed a similar pH dependence in 4 M Li ϩ and in 4 M Na ϩ (Fig+ 2B)+ For both monovalent and divalent cations, it appears there is a single deprotonation prior to the ratelimiting step, most likely deprotonation of the 29-OH at the site of cleavage (Dahm et al+, 1993;Kuimelis & McLaughlin, 1995; but also see Sawata et al+, 1995)+ Throughout this pH range, deprotonation of the 29-OH might be only about sevenfold greater in Mg 2ϩ than in Li ϩ +…”

Inhibition of the Hammerhead Ribozyme Cleavage Reaction by Site-Specific Binding of Tb(III)

“…Biochemical methods available to evaluate the role of these metal ions in ribozyme function are limited. The most common approach is to introduce a phosphorothioate modification into the RNA and to examine its effect on the metal specificity of the catalytic reaction (8)(9)(10)(11)(12)(13). We present an approach to studying metal binding to ribozymes based on the observation that ions that compete efficiently for critical Mg-binding sites can thereby inhibit catalysis.…”