Activation of d-Tyrosine by Bacillus stearothermophilus Tyrosyl-tRNA Synthetase

Tyrosyl-tRNA synthetase (TyrRS) is able to catalyze the transfer of both l- and d-tyrosine to the 3′ end of tRNATyr. Activation of either stereoisomer by ATP results in formation of an enzyme-bound tyrosyl-adenylate intermediate and is accompanied by a blue shift in the intrinsic fluorescence of the protein. Single turnover kinetics for the aminoacylation of tRNATyr by d-tyrosine were monitored using stopped-flow fluorescence spectroscopy. Bacillus stearothermophilus tyrosyl-tRNA synthetase binds d-tyrosine with an 8.5-fold lower affinity than that of l-tyrosine (KD-Tyrd = 102 μm) and exhibits a 3-fold decrease in the forward rate constant for the activation reaction (kD-Tyr3 = 13 s–1). Furthermore, as is the case for l-tyrosine, tyrosyl-tRNA synthetase exhibits “half-of-the-sites” reactivity with respect to the binding and activation of d-tyrosine. Surprisingly, pyrophosphate binds to the TyrRS·d-Tyr-AMP intermediate with a 14-fold higher affinity than it binds to the TyrRS·l-Tyr-AMP intermediate (KPPid = 0.043 for TyrRS·d-Tyr-AMP·PPi). tRNATyr binds with a slightly (2.3-fold) lower affinity to the TyrRS·d-Tyr-AMP intermediate than it does to the TyrRS·l-Tyr-AMP intermediate. The observation that the KTyrd and k3 values are similar for l- and d-tyrosine suggests that their side chains bind to tyrosyl-tRNA synthetase in similar orientations and that at least one of the carboxylate oxygen atoms in d-tyrosine is properly positioned for attack on the α-phosphate of ATP.