282 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
P1.1 pro - R p phosphoryl oxygen – sulfur substitution consistent with Mg 2+ inter-
actions seen in the Scott crystal structure (PDB: 301D) obtained at pH 8.5,
because the preceding NMR experiments were conducted at this pH. In the
same NMR experiment conducted at pH 5.5, no Cd 2+ shifts of the^31 P NMR
spectra were seen for the substrate P1.1 pro - R p phosphoryl oxygen – sulfur
substitution.
Comparison of the described results for the hammerhead complex to those
obtained with several model compounds led to the conclusions that the upfi eld
(coordination to sulfur) or downfi eld (coordination to oxygen) shifts could be
noted for the model compounds as well. Similar NMR results were obtained
whether a DNA or an RNA – OMe substrate was used in conjunction with the
hammerhead ribozyme RNA, and substitution of sulfur at other phosphate
oxygen positions within the ribozyme or substrate did not cause binding of
Cd2+ or^31 P NMR shifts. Comparison of the NMR results with those obtained
previously using Mn 2+ in EPR, ENDOR, and ESEEM studies^46 showed that
there is at least one high - affi nity metal binding site ( Kd ≤ 10 micromolar, μ M)
and at least one, and possibly more, low - affi nity sites since full hammerhead
activity requires > 1 millimolar, mM Mn 2+ (or Mg 2+ as determined by other
experimentalists). All observations taken together indicate that the A9/G101.1
Figure 6.19 The pro - R p oxygen atoms at two sites in the PDB: 1HMH hammerhead
ribozyme. (A) The Cleavage site (P1.1) and (B) the A9/G10.1 site. (Adapted with per-
mission from Figure 1 of reference 20. Copyright 2000, American Chemical Society.)
Visualized using CambridgeSoft Chem3D Ultra 10.0 with notations in ChemDraw
Ultra 10.0 (Printed with permission of CambridgeSoft Corporation.)
C17G1.1 pro-RpO5’ O G1.13’ O C17A9A
Cleavage site (P1.1)pro-Rp O3’ O G8B
A9/G10.1 site5’ O A9G10.1G8