STEREOCHEMISTRY OF TRNA(M(5)U54)-METHYLTRANSFERASE CATALYSIS - F-19 NMR-SPECTROSCOPY OF AN ENZYME-FURARNA COVALENT COMPLEX

The catalytic mechanism of tRNA(m(5)U54)-methyltransferase (RUMT) invo lves the formation of a covalent adduct between Cys324 of RUMT and C6 of Ura54 in tRNA. The covalent adduct is subsequently methylated at C5 by S-adenosyl-L-methionine (AdoMet). We used an RNA substrate analog containing 5-fluorouracil (FUra) in place of Ura54 to trap the covalen t complex and analyzed the adduct by F-19 NMR spectroscopy. The F-19 N MR spectrum of the adduct consisted of an overlapping doublet of quart ets, with an H-6-F coupling constant of 4 Hz and a CH3-F coupling cons tant of 22.4 Hz. On the basis of the magnitude of the H-6-F coupling c onstant, we determined that Cys324 of RUMT and the methyl moiety from AdoMet added across the 5,6-double bond of FUra54 in cis fashion. We d educed that the nucleophilic addition was also cis in the normal enzym atic reaction and that the subsequent beta-elimination of the 5-H and catalytic cysteine was trans. Further, on the basis of chemical consid erations, we proposed several conformational adaptations of enzyme-sub strate complexes that must occur on the reaction pathway. Together wit h previous studies, this study enables the proposal of the complete st ereochemical pathway for the RUMT-catalyzed methylation of Ura54 in tR NA.