A CONSERVED ASPARTATE OF TRANSFER-RNA PSEUDOURIDINE SYNTHASE IS ESSENTIAL FOR ACTIVITY AND A PROBABLE NUCLEOPHILIC CATALYST

tRNA pseudouridine synthase I catalyzes the conversion of uridine to p seudouridine at positions 35, 39, and/or 40 in the anticodon loop of m any tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter an d expressed in Escherichia coli to about 20% of total soluble proteins , Fluorouracil-substituted tRNA caused a time-dependent inactivation o f pseudouridine synthase I and formed a covalent complex with the enzy me that involved the FUMP at position 39. Asp60, conserved in all know n and putative pseudouridine synthases, was mutated to amino acids wit h diverse side chains. All Asp60 mutants bound tRNA but were catalytic ally inactive and failed to form covalent complexes with fluorouracil- substituted tRNA. We conclude that the conserved Asp60 is essential fo r pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.