Interaction of tRNA (uracil-5-)-methyltransferase with NO2Ura-tRNA.
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.
Gu
X
X
Department of Biochemistry, University of California, San Francisco 94143-0448 USA.
Matsuda
A
A
Ivanetich
K M
KM
Santi
D V
DV
D001665:Binding Sites; D001667:Binding, Competitive; D003101:Collodion; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012359:tRNA Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D046911:Macromolecular Substances; D012329:RNA, Bacterial; D014498:Uracil; C057449:5-nitrouracil; D003101:Collodion; D012343:RNA, Transfer; D012359:tRNA Methyltransferases; C022769:tRNA(uracil-5)-methyltransferase