The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (Psi SI) catalyzes the conversion of uridine to Psi at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. Psi S I forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA(Phe) contai ning FUra in place of Ura) to form a putative analog of a steady-state inte rmediate in the normal reaction pathway. Previously, we proposed that a con served aspartate of the enzyme serves as a nucleophilic catalyst in both th e normal enzyme reaction and in the formation of a covalent complex with FU ra-tRNA. The covalent adduct between FUra-tRNA and Psi SI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5 ,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluor ouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming t hat an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for Psi S.