Cobalt hexammine inhibition of the hammerhead ribozyme.
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.
Horton
T E
TE
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, USA.
DeRose
V J
VJ
D001667:Binding, Competitive / Q000187:drug effects; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D008345:Manganese / Q000737:chemistry; D008667:Metalloproteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D009691:Nucleic Acid Denaturation / Q000187:drug effects; D016337:RNA, Catalytic / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D013696:Temperature
D004791:Enzyme Inhibitors; D008667:Metalloproteins; D016337:RNA, Catalytic; C032300:hexaamminecobalt(II); D003035:Cobalt; D008345:Manganese