Activation of D-tyrosine by Bacillus stearothermophilus tyrosyl-tRNA synthetase: 1. Pre-steady-state kinetic analysis reveals the mechanistic basis for the recognition of D-tyrosine.
Tyrosyl-tRNA synthetase (TyrRS) is able to catalyze the transfer of both l- and d-tyrosine to the 3' end of tRNA(Tyr). 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 tRNA(Tyr) 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 (K (D-Tyr)(d) = 102 microm) and exhibits a 3-fold decrease in the forward rate constant for the activation reaction (k (D-Tyr)(3) = 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 (K (PPi)(d) = 0.043 for TyrRS.d-Tyr-AMP.PP(i)). tRNA(Tyr) 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 K (Tyr)(d) and k(3) 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 alpha-phosphate of ATP.
Sheoran
Anita
A
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, USA.
Sharma
Gyanesh
G
First
Eric A
EA
D049148:Aminoacylation; D011756:Diphosphates / Q000378:metabolism; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000378:metabolism*
D011756:Diphosphates; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase