272 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
in position is accompanied by other nucleobase and ribose movements with
concomitant changes in hydrogen bonding patterns in the region. Signifi cant
new hydrogen bonds stabilizing the conformational change included one that
formed between the furanose oxygen (O4 ′ ) of C 17.0 and the 2O ′ of U 16.1 ’ s 2 ′ -
hydroxyl group. Importantly, a magnesium ion (site 6) appears to be approxi-
mately 2.4 Å from the pro - R p phosphate oxygen of residue A 1.1. Another
magnesium ion (site 4) appears near to G 5.0 ; this site is known to be required
for hammerhead catalytic activity. A third magnesium ion (site 2) appears near
the A 9.0 – G 10.1 region but does not interact in the manner seen for the manga-
nese ion in the PDB: 300D structure. The site 6 magnesium ion may be induc-
ing a conformational change leading to transition state in which in - line S N 2
attack could occur. Although it is not possible to visualize the coordination
sphere of the magnesium ion at the resolution of the crystal structures (3.00 Å ),
the reference 39 authors hypothesized that a coordinated hydroxide could
provide the catalyst for the base - catalyzed step of the cleavage reaction. They
also favored a mechanism in which the Mg 2+ – phosphate complex rotated rela-
tive to the rest of the RNA molecule to reach a transition state that would
bring the O2 ′ of C 17.0 ’ s 2 ′ - OH group in line for nucleophilic attack on the
phosphate phosphorus atom. (O ′ indicate ribose oxygen atoms). Herschlag ’ s
group, reference 38 , commented that the observed PDB: 301D structure might
represent an early intermediate on the reaction pathway, but differing consid-
erably from the transition state structure, or might be an “ off - pathway ” struc-
ture. If crystals of the Mg 2+ - modifi ed ribozyme – substrate complex (PDB:
301D) are dissolved after being soaked in 100 mM MgSO 4 at pH 8.5 for 15
minutes, a denaturing polyacrylamide gel assay shows very little residual intact
25 - nt substrate and a substantial amount of 20 - nt cleaved product (see Figure
2 of reference 39 ).
In 1997, Scott and co - workers provided evidence gathered in molecular
dynamic simulations that a hydroxide ion bridge could exist between two
magnesium ions near the hammerhead cleavage site.^40 Starting with data
from the X - ray crystallographic structure PDB: 301D described in the previous
paragraphs, the μ - bridging hydroxide ion was located, in the simulations,
between site 6 (that interacts with a nonbridging phosphate oxygen at the
cleavage site) and site 1 magnesium ions. Using numbering provided with the
Protein Data Bank fi le for PDB: 301D, one fi nds that the site 1 – site 6 dis-
tance — Mg(876) – Mg(880) — is equal to 4.46 Å. Further simulations showed that
fl ipping the ribose at the cleavable phosphate site from the C3 ′ - endo to the
2 ′ - endo conformation brings the relevant 2 ′ - OH nucleophilic group in proxim-
ity to the attacked phosphate phosphorus atom and the μ - bridging OH − ion.
Herschlag ’ s group continued its study of structure – function relationships in
the hammerhead ribozyme using a “ base - rescue ” biochemical method. This
method substitutes other atoms or molecules for bases at critical catalytic
or structural positions and tests whether catalytic activity is lost. If so, the
RNA bases (U, A, G, C) or a modifi ed base (for instance, deazaguanine or 2 -
aminopurine substituted for guanine) is added to the solution to ascertain