Bacterial tRNA-specific adenosine deaminase (TadA) catalyzes the essential deamination of adenosine to inosine at the wobble position of tRNAs and is necessary to permit a single TRNA species to recognize multiple codons. The transition state structure of Escherichia coli TadA was characterized by kinetic isotope effects (KIEs) and quantum chemical calculations. A stem loop of E. coli tRNAArg2 was used as a minimized TadA substrate, and its adenylate editing site was isotopically labeled as [1′-3H], [5′-3H2], [1′-14C], [6- 13C], [6-15N], [6-13C, 6-15N] and [1-15N]. The intrinsic KIEs of 1.014, 1.022, 0.994, 1.014 and 0.963 were obtained for [6-13C]-, [6-15N]-, [1-15N]-, [1′-3H]-, [5′-3H2]-labeled substrates, respectively. The suite of KIEs are consistent with a late S NAr transition state with a complete, pro-S-face hydroxyl attack and nearly complete N1 protonation. A significant N6-C6 dissociation at the transition state of TadA is indicated by the large [6-15N] KIE of 1.022 and corresponds to an N6-C6 distance of 2.0 Å in the transition state structure. Another remarkable feature of the E. coli TadA transition state structure is the Glu70-mediated, partial proton transfer from the hydroxyl nucleophile to the N6 leaving group. KIEs correspond to H-O and H-N distances of 2.02 and 1.60 Å, respectively. The large inverse [5′-3H] KIE of -3.7% and modest normal [1′-3H] KIE of 1.4% indicate that significant ribosyl 5′-reconfiguration and purine rotation occur on the path to the transition state. The late SNAr transition-state established here for E. coli TadA is similar to the late transition state reported for cytidine deaminase. It differs from the early SNAr transition states described recently for the adenosine deaminases from human, bovine, and Plasmodium falciparum sources. The ecTadA transition state structure reveals the detailed architecture for enzymatic catalysis. This approach should be readily transferable for transition state characterization of other RNA editing enzymes.
ASJC Scopus subject areas
- Colloid and Surface Chemistry