Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermoph ilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha -ammonium group of the tyrosine substrat e during the first step of the aminoacylation reaction. Asp194 and Gln195 s tabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxy gen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln 173, Asp194, and Gln195 play in catalysis of the second step of the reactio n are investigated. Pre-steady-state kinetic analyses of alanine variants a t each of these positions shows that while the replacement of Gln173 by ala nine does not affect the initial binding of the tRNA(Tyr) substrate, it des tabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transit ion state for the second step of the aminoacylation reaction. Taken togethe r, the results presented here and the accompanying paper are consistent wit h a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition stab in which the scissile acylphosp hate bond of the tyrosyl-adenylate species is strained. Cleavage of the sci ssile bond on the breakdown of the transition state alleviates this strain. (C) 2000 Academic Press.