Chemistry Reference
In-Depth Information
When
N
-acetylphenylalanyl-tRNA containing 8-azidoadenosine in the 73 position
was bound to the P-site using the codon provided by polyU, and irradiated, the 23S
ribosomal RNA was the main ribosomal component cross-linked.
145
This study
provides an example of the dynamics of enzyme action and how the enzyme
geometry changes as the programmed reaction progresses. The tRNA enters the
P-site with an attached amino acid and the ribosome folds to accommodate this
situation, which apparently brings 23S ribosomal RNA into close proximity to the
tRNA
Phe
3
0
terminus. Following transfer of the growing peptide chain to the tRNA in
the entrance or A-site, the unacylated tRNA
Phe
occupies the P-site, and ribosome
refolds and brings a portion the 50S subunit close to the tRNA
Phe
3
0
terminus. In this
way, photoaffinity labeling can provide insights into the dynamics of biological
processes that cannot be obtain even in an X-ray structure where the system is frozen
in a single geometry.
In many PAL applications, a molecule with a single photoactivatable unit is
employed, and this PAL unit is attached to another unit that has an affinity for a
particular site or molecule of interest. Molecules of this type are applied to “map” the
molecular environment of the target site. In the azidoadenosine strategies outlined
above, the affinity units and the PAL units have been integrated into the same
molecule. However, for mapping proteins made up from several subunits, each of
which has a binding site for ATP, it is useful to apply molecules with two separate
PAL units built into the same affinity agent. The diPAL reagent
(Scheme 3.29) has
been successfully used in the study of phosphofructokinase-1 that is composed of
eight subunits, four
47
-subunits. Each of these subunits has
regulatory binding sites for MgATP. When phosphofructokinase-1 was treated with
a
-subunits and four
b
N
NH
2
N
N
N
N
N
3
N
3
N
N
O
O
N
O
O
N
O
P
O
-
O
P
O
P
O
P
O
-
O
O
O
O
-
O
-
OH
OH
OH
OH
47
NH
2
N
NH
2
N
N
3
N
N
N
N
3
O
N
N
3
-
O
P
O
-
O
N
O
O
O
N
O
-
O
P
O
P
O
P
O
-
O
O
OH
OH
O
-
O
-
O
2
N
OH
O
49
48
CH
2
-CH
2
-NH
N
3
O
SCHEME 3.29.
