Chemistry Letters 2001
1071
On the other hand, the influence of TC was investigated for
TD reaction. Although TC prepared without histidine did not
show notable influence on the TD reaction products, the yield of
oligo(G) was fairly reduced at 0.06 M TC-his, in which the con-
centrations of TC were calculated on the basis of amino acid unit
(Table 2). Further, the extent of P1,P2-bis(5'-guanosyl) diphos-
phate (G5'ppG) was also increased with TC-his. To demonstrate
more clearly the inhibition by TC, TC-std and TC-his were dia-
lyzed and then used for the TD reaction. Table 2 indicates that the
formation of oligo(G) was inhibited and the hydrolysis of
2MeImpG was enhanced at 0.06 M TC-his dialyzed using either
MWCO 1000 or 12000–14000 tubing, while TC-std changed less
the yield of oligo(G).
The catalytic ability of histidine and TC-his for the
2MeImpG hydrolysis is unexpected finding since it has been
observed in previous kinetic studies that simple bases, such as
imidazole and 2-methylimidazole, do not have catalytic ability
for the hydrolysis of activated nucleotides.8 Further, the results
in the present study indicate that histidine incorporated in TC is
essential for the catalysis of 2MeImpG hydrolysis, where the
histidine residues would be important. Thus, details how histi-
dine is incorporated as the catalytic site of TC-his will be help-
ful to understand the reaction mechanism in future.
We thank Professor Taketoshi Nakahara of Osaka
Prefecture University for the use of HPLC apparatus. This
research was partially supported by the Sumitomo Foundation
1998, Japan.
On the other hand, the extent of G5'ppG considerably
increased in the presence of TC (Table 2). Further, HPLC chro-
matogram of TD reaction at 0.05 M TC-std dialyzed with
MWCO 12000–14000 tubing shows additional peaks (Figure 2).
Besides, it is known that G5'ppG-capped oligo(G) forms from TD
reaction under several conditions.9 Thus, these facts indicate that
the formation of G5'ppG-capped oligo(G) may contribute to the
inhibition of TD reaction by TC. To evaluate this assumption,
the TD reaction with 0.0015 M G5'ppG in the absence of TC was
carried out, in which the TD reaction products showed very simi-
lar HPLC pattern to that observed in the presence of TC. This
fact suggests that oligo(G) formed with TC-std and TC-his
involves G5'ppG-capped oligo(G) isomers (Figure 3).
It is widely believed that amino acids formed abundantly on
the primitive earth.4 Besides, there are hypotheses that primitive
proteins were constructed from only 4 kinds of amino acids, that
is, glycine, alanine, valine, and aspartic acid, so TC used in this
study may resemble to primitive enzymes in the compositions of
amino acids.10 Thus, finding of inhibition by TC-his and histi-
dine itself for the TD reaction may oppose to the RNA world
hypothesis. Further investigations on the cooperative chemical
evolution of nucleic acids and proteins are being in progress.
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