| Literature DB >> 8604339 |
X Gu1, A Matsuda, K M Ivanetich, D V Santi.
Abstract
tRNA in which uracil is completely replaced by 5-nitro-uracil was prepared by substituting 5-nitro-UTP for UTP in an in vitro transcription reaction. The rationale was that the 5-nitro substituent activates the 6-carbon of the Ura heterocycle towards nucleophiles, and hence could provide mechanism-based inhibitors of enzymes which utilize this feature in their catalytic mechanism. When assayed shortly after mixing, the tRNA analog, NO2Ura-tRNA, is a potent competitive inhibitor of tRNA-Ura methyl transferase (RUMT). Upon incubation, the analog causes a time-dependent inactivation of RUMT which could be reversed by dilution into a large excess of tRNA substrate. Covalent RUMT-NO2Ura-tRNA complexes could be isolated on nitrocellulose filters or by SDS-PAGE. The interaction of RUMT and NO2Ura-tRNA was deduced to involve formation of a reversible complex, followed by formation of a reversible covalent complex in which Cys 324 of RUMT is linked to the 6-position of NO2Ura 54 in NO2Ura-tRNA.Entities:
Mesh：
Substances：
Year: 1996 PMID： 8604339 PMCID： PMC145746 DOI： 10.1093/nar/24.6.1059
Source DB: PubMed Journal: Nucleic Acids Res ISSN： 0305-1048 Impact factor: 16.971