Cobalt Hexammine Inhibition of the Hammerhead Ribozyme

Abstract

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.

The RNA‐Cleaving Bipartite DNAzyme Is a Distinctive Metalloenzyme

“…The slope of the Hill plot was À1.44 AE 0.30. Given that a hexaamminecobalt(III) cation is able to substitute one-to-one for a structurally isomorphous Mg 2 + or Mn 2 + ion, [24] this inhibition slope is consistent with both mono-and dicoordinated manganese ion complexes being catalytically active. The reverse titration was also carried out, in which in the presence of a fixed hexaamminecobalt concentration of 0.5 mM k obs values were measured with progressive titrations of manganese (data not shown).…”

“…[24] Since the hexaamminecobalt(III) cation is substitution-inert with respect to its ammonia ligands (and given that the metalbound ammonia ligands are incapable of the acid-base chemistry characteristic of metal-bound water or hydroxide ligands), competition experiments involving titration with hexaamminecobalt(III) salts into reaction solutions containing a fixed Mg 2 + (or Mn 2 + ) concentration (and vice versa) are able to reveal the catalytic necessity-or not-of Mg 2 + or Mn 2 + . Our competition experiments, carried out on the manganese-utilizing DNAzyme, revealed that the hexaamminecobalt-(III) cation inhibited the RNA cleavage reaction.…”

Distinct reaction pathway promoted by non-divalent-metal cations in a tertiary stabilized hammerhead ribozyme

“…3A) are significantly lower than those seen in Mg 2+ or other divalent ions at >90% or in sodium or spermidine at 70%-80% ( Fig. 2B ions, with accompanying structural changes, has also been observed in a minimal hammerhead ribozyme (Horton and DeRose 2000).…”

“…In the Neurospora crassa VS ribozyme, it enhances activity in the presence of Mg 2+ but cannot support catalysis as the sole metal ion (Maguire and Collins 2001). In the HDV ribozyme (Nakano et al 2000) and in a minimal hammerhead ribozyme (Horton and DeRose 2000), CoHex inhibits activity in the presence of other divalent ions, suggesting a competition for binding. Previous studies with minimal hammerhead ribozymes showed very slow cleavage activity (z0.0071 min…”

Mechanistic studies on acyl‐transferase ribozymes and beyond

“…29 These effects have been seen in many ribozyme systems and have been attributed to the excess metal ions causing distortions of ribozyme structure. 41 Monovalent metal ions have also been discussed in the context of ribozyme catalysis. 42 The selection of ATRib (E18) without offering the same catalytic effect.…”