Chemistry Reference
In-Depth Information
NH
2
N
N
N
a
N
NH
2
N
N
N
N
R
N
NH
2
N
N
3
N
N
N
R
b
N
N
N
R
N
N
SCHEME 3.28.
In spite of these potential complications, derivatives of 2-azidoadenosine have
been used successfully in several biological environments. In one such application,
the ADP mimic
30
was used to study the structure of rabbit skeletal myosin, see
above. However,
resembles ADP only in that it contains a diphosphate extension.
Therefore, this work has been repeated using 2-azidoadenosine diphosphate to afford
18-14% cross-linking to the same Trp
130
30
,
143
residue observed with
30
thus,
reinforcing the previous results.
83-88
Some proteins are not synthesized by the usual DNA/mRNA/tRNA/ribosome
route, but by specific enzyme systems. One such enzyme is tyrocidine synthetase that
catalyzes the first step in the synthesis of the decapeptide antibiotic tyrocidine. This
process has an ATP requirement and has been studied using both 8N
3
ATP and
2N
3
ATP.
144
Apparently, 8N
3
ATP is not accepted into the active site of this enzyme,
but 2N
3
ATP is accepted, and successfully photolabels the enzyme. Three tryptic
peptides were isolated and characterized from the
N
-terminal half of the enzyme.
Apparently, 8N
3
ATP assumes the unacceptable syn-conformation.
138
and therefore,
is not bound to the synthetase active site, but 2N
3
ATP retains the acceptable anti-
conformation
139
and is bound to the active site.
This same syn/anti-problem also apparently comes into play in many of the
labeling strategies involving 8-azidoadenosine. Both [5
0
-
32
P]p8N
3
Ap and [5
0
-
32
P]
p2N
3
Ap have been used to study the binding of tRNA
Phe
to the ribosome. The
3
0
-terminal nucleotides of tRNA
Phe
are -A
73
-C
74
-C
75
-A
76
(see Fig. 3.5 for reference
to tRNA structure). Both of these azido diphosphates indicated earlier have been
used to replace the adenosines in the 73 and 76 positions. If the 8-azido diphosphate
was located in position 76, aminoacylation was completely inhibited, but if it was
located in the 73 position, aminoacylation occurred normally.
145
Alternatively, the
2-azido diphosphate could be located in the 76 position, and aminoacylation
proceeded normally.
146
When either p8N
3
Ap or p2N
3
Ap were inserted into tRNA
Phe
at the 73 and 76 positions, respectively, and irradiated in the ribosome in the presence
of the appropriate mRNA (polyU), which positioned the tRNA
Phe
in the middle or
P-site of the ribosome, the 50S subunit of the ribosome was covalently cross-linked.
