4.6 Article

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

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JOURNAL OF BIOLOGICAL CHEMISTRY
卷 283, 期 19, 页码 12960-12970

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AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M801649200

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  1. NIGMS NIH HHS [GM68070, R01 GM068070] Funding Source: Medline

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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 (K-d (D-Tyr) = 102 mu M) and exhibits a 3-fold decrease in the forward rate constant for the activation reaction (k(3)(D-Tyr) = 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-TyrAMP intermediate (K-d(PPi) = 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.LTyr-AMP intermediate. The observation that the K-d(Tyr) 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.

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