Revised Manuscript Received August 14, 1995® abstract: A series of 5-substituted 2-aminopyrrolo [2, 3-< i] pyrimidin-4 (3//)-ones have been synthesized in order to study the substrate specificity of the tRNA-guanine transglycosylase (TGT) from Escherichia coli. A number of these compounds were initially examined as inhibitors of radiolabeled guanine incorporation into tRNA catalyzed by TGT [Hoops, G. C., Garcia, G. A., & Townsend, L. B.(1992) 204th National Meeting of the American Chemical Society, Washington, DC, August 23—28, 1992, Division of Medicinal Chemistry, Abstract 113]. The kinetic parameters of these analogues as substrates in the TGT reaction have been determined by monitoring the loss of radiolabeled guanine from 8-[14C] G34-tRNA. This study reveals that the tRNA-guanine transglycosylase from E. coli will tolerate a wide variety of substituents at the 5-position. The role of the 5-substituent appears to be entirely in binding/recognition with no apparent effects upon catalysis. A correlation between N7 pA" a and Vmax suggests the deprotonation of N7 during the reaction, which must occur prior to subsequent glycosidic bond formation, appears to be partially rate-determining for the natural substrate. Comparison of the K\s of 7-methyl-substituted competitive inhibitors to the A'mS of their corresponding substrates suggests that some substrates (including preQi) are kinetically “sticky”{ie, Km is equivalent to K¿) and other substrates have Kms that reflect catalytic rates as well as binding.

To date, over 90 modified nucleosides have been discov-ered in RNA, over 70 in transfer RNA alone (Limbach et al., 1994). In many cases their stmctures, positions in specific tRNAs, and biosynthetic pathways havebeen elucidated. However, relativelylittle is known about their biological roles and the molecular mechanisms by which those roles are performed. One of the hypermodified nucleoside bases found in tRNA is the guaninederivative queuine [2-amino-5-[[(4, 5-cri-dihydroxy-l-cyclopenten-3-yl) amino] methyl] pyrrolo [2, 3-£ f| pyrimidin-4 (3//)-one, 1, Fig-ure 1], This is the first modified base in which the basic (parent) ring system (purine or pyrimidine) has been modified. tRNA-guanine transglycosylase (TGT)* 1 is the enzyme responsible for the post-transcriptional modification of specific tRNAs (Tyr, His, Asp, and Asn) with queuine. There appear to be two functionally different classes of the