Studies of the heterocyclic substrate for thetRNA-guanine transglycosylase from Escherichia coli.

Abstract

A series of 5-substituted 2-aminopyrrolo(2,3-d) pyrimidin-4-ones were studied in order to determine the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli. The kinetic parameters of these analogues as substrates in the TGT reaction were determined by monitoring the loss of 8- ($\sp{14}$C) -guanine from G34-tRNA. These studies indicated that TGT tolerates a wide range of substitutions at the 5 position. Compounds containing 5-substituents which are electron-withdrawing exhibited higher $V\sb{max}$ values, suggesting that deprotonation at N-7 may be partially rate limiting in the TGT reaction. The 3-methyl and 6-methyl analogues are inactive. A number of 7-methyl-substituted analogues were found to act as competitive inhibitors of the TGT reaction. The $K\sb{I}$'s for two of these analogues are in good agreement with the $K\sb{M}$'s for the corresponding substrates, however, the $K\sb{I}$ for the third analogue is significantly higher than the $K\sb{M}$ for its corresponding substrate. This suggests that some substrates (including preQ$\sb1$) are kinetically sticky (i.e., $K\sb{M}$ is equivalent to $K\sb{D}$) and other substrates have $K\sb{M}$'s that reflect catalytic rates as well as binding. The deprotonation of N-7 of the heterocyclic substrate was examined in more detail by synthesizing a series of N-3- and N-7-methylated analogues of those compounds studied an Chapter 2. These studies indicated that N-7 of those analogues bearing an electron-donating 5-substituent (including preQ$\sb1$) have a pK$\rm\sb{a} > 13.$ Thus enzymatic activation is required for the deprotonation of N-7. The finding that deprotonation of N-7 of 2-aminopyrrolo (2,3-d) pyrimidin-4-one substrates for TGT appears to be at least partially rate limiting combined with the knowledge that this process occurs with a pK$\rm\sb{a} > 13$ led to the design and synthesis of a series of 5-halomethyl 2-aminopyrrolo (2,3-d) pyrimidin-4-ones (81-84) as potential mechanism-based inhibitors of TGT (Chapter 4). Compounds 81-84 were all found to be time-dependant inhibitors of TGT in preincubation experiments. The inclusion of a pyrrole-methylated analogue of preQ$\sb1$ at saturating concentrations in this assay mixture was found to protect the enzyme from inactivation. The inactivated enzyme was passed through a gel filtration column and assayed for activity. No activity was reconstituted following desalting, indicating that the time-dependant inactivation is irreversible. The inactivated enzyme, before and after desalting, remained identical to the active enzyme by SDS-PAGE and native-PAGE. Compound 81, the most active irreversible inhibitor of the series, was selected for further studies. This compound partitioned between halide release and inactivation of enzyme, indicating that its inactivation of TGT was mechanism-based. A series of 5-haloacetyl 2-aminopyrrolo (2,3-d) pyrimidin-4-ones was prepared (Chapter 5) as potential active-site-directed irreversible inhibitors. However these compounds appear to be completely devoid of activity with TGT.