期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1999
卷号：96
期号：25
页码：14270-14275
DOI：10.1073/pnas.96.25.14270
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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}SI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-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-fluorouridine. 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 that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for {Psi}S.
