MAGNESIUM AND CATALYTIC RNA 243
and transition states. Therefore, one might have an active site in one conforma-
tion that uses metal ion cofactor MgA^2 +, while another conformation uses metal
ion cofactor MgB^2 + in its active site. DeRose, in reference 16 , suggests that a
metal ion may be considered to be functionally involved in catalysis if it (1)
contributes to stabilizing the transition state and (2) is associated with a group
that changes bond order during the reaction. Conversely, if the metal ion can
be predicted to be far (and stay far) from the active site, then it should be
considered as playing only a structural role. It should also be remembered that
sulfur atoms are larger and more polarizable than oxygen atoms and that they
may perturb ribozyme structure, in some cases to an unacceptable degree. In
one case, that of the hammerhead ribozyme, to be discussed in more detail
below, PS - rescue experiments have been confi rmed using^31 P NMR.^20 Electron
paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR),
and electron spin - echo envelope modulation (ESEEM) are other spectro-
scopic methods used successfully to determine metal ion occupation at the
active site in the hammerhead ribozyme.^21 (See Section 3.5 for more informa-
tion on EPR techniques and see Section 3.5.3 for more information on ENDOR
and ESEEM.) Use of EPR requires that a paramagnetic ion take the place of
magnesium ions in the ribozyme. Fortunately, the paramagnetic Mn 2+ ion is
similar in ionic radius, enthalpy of hydration, and rate of catalytic activity
when substituted for magnesium ions in the hammerhead ribozyme or in the
group I intron. Hyperfi ne interactions between the Mn 2+ electron spin and
nuclei of the RNA ligands are observed using ENDOR and ESEEM. These
help identify Mn 2+ ions directly coordinated to phosphate and aqua ligands.
These results for the hammerhead ribozyme will be discussed further in Section
6.2.4. Conclusions from the reference 16 review paper include the following:
(1) Separating cation infl uences on RNA structure from those on catalytic
activity constitutes a signifi cant experimental challenge; (2) high - and low -
affi nity cation interactions have been identifi ed as being required for ribozyme
activity (high - affi nity sites are involved in folding while low - affi nity sites are
involved in catalysis); and (3) although cations infl uence ribozyme function,
the majority of catalytic power is probably derived from the nucleobases
themselves.
In discussing the phosphorothioate metal - rescue (PS - rescue) experiment in
the previous paragraph, it was mentioned that replacing one of the phosphate
oxygens in an RNA polymer with a sulfur atom produces a chiral center. We
say that the phosphorus atom in the phosphate connector is pro - chiral because
changing one of two oxygen atoms (substituting a sulfur for instance as in the
P - S rescue experiment) gives rise to a chiral center. Replacing one specifi c
oxygen with a sulfur will give rise to an S confi guration (pro - S oxygen), while
changing the other one would yield an R confi guration (pro - R oxygen) accord-
ing to Cahn – Ingold – Prelog rules. For a phosphate group in an RNA chain, in
which one goes toward the 5 ′ - carbon atom of a sugar moiety in one direction
and towards the 3 ′ - end of another sugar in the opposite direction, the proce-
dure for identifying pro - R and pro - S oxygens is outlined in Figure 6.2. Identi-