PMID:9425056
| Citation |
Huang, L, Pookanjanatavip, M, Gu, X and Santi, DV (1998) A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. Biochemistry 37:344-51 |
|---|---|
| Abstract |
tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles. |
| Links |
PubMed Online version:10.1021/bi971874+ |
| Keywords |
Amino Acid Sequence; Aspartic Acid/genetics; Aspartic Acid/metabolism; Binding Sites/genetics; Catalysis; Cloning, Molecular; Conserved Sequence; Electrophoresis, Polyacrylamide Gel; Enzyme Activation/genetics; Escherichia coli; Fluorouracil/pharmacology; Genetic Vectors/metabolism; Intramolecular Transferases/antagonists & inhibitors; Intramolecular Transferases/genetics; Intramolecular Transferases/metabolism; Molecular Sequence Data; Mutagenesis; Phosphorus Radioisotopes; RNA, Transfer, Asp/genetics; RNA, Transfer, Asp/metabolism; RNA, Transfer, Phe/pharmacology; Uracil/pharmacology |
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