Biology Reference
In-Depth Information
loss of catalytic activity.
83,94,95
Nevertheless, these mutational effects cannot
be explained from the mHHR structure based on the interruption of key
indices, such as Watson-Crick hydrogen bond interactions.
61
2.1.3 Lack of structural support for general acid and base candidates
identified from biochemical experiments
It has been recently suggested that the conserved residues G8 and G12 act as
the general acid and the general base, respectively, of the HHR reaction
mechanism.
96
However, it was not clear from available crystallographic data
how these two residues could play such roles given they are distant from the
cleavage site in the mHHR structure.
2.1.4 Other concerns with the mHHR structure
NMR data and chemical mutation data suggest that U4 and U7 bases must
approach one another within about 6
˚
, although it did not appear to be
possible from the mHHR crystal structure.
97,98
Furthermore, in all publi-
shed mHHR structure, none has an in-line attack angle near 180 degrees,
which is required by the first nucleophilic attacking step of the HHR
reaction.
2.2. The crystal structures of the full-length (extended) HHR
The long-standing debate between structural and biochemical data of the
mHHR was finally resolved with the determination of the first crystal struc-
ture of the eHHR (63 residues) by Martick and Scott,
73
and elucidation of
the details of its solvent structure.
74
The eHHR crystal structure indicates a
significant rearrangement from the mHHR structures that allow extensive
new interactions between the loops of stem II and stem I, and almost all dis-
crepancies are resolved in this structure. The distance between the oxygen
atoms (A9:O2P and C1.1:O2P) is around 4
˚
, C3 and G8 form a canonical
Watson-Crick pair, G5 holds hydrogen bonds interacting with the active
site (C1.1 and C17), G8 and G12 are in the ready positions as the general
acid/base, U4 and U7 are nearby, and the nucleophile (C17:O2
0
) has an
in-line attacking angle around 160 degrees.
68,99-101
The existing mutagen-
esis data also agree well with this eHHR crystal structure.
68
The eHHR structure successfully resolved a wealth of problems regard-
ing the basic architecture of the active site that had been points of (often
heated) contention. However, it also brought a new dimension to the prob-
lem regarding the identification of the role of divalent metal ions which,
under physiological ionic strength conditions, were known to be critical
Search WWH ::
Custom Search