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. 2012 May 30;287(30):25381–25394. doi: 10.1074/jbc.M112.372151

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© 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 6. — Transient formation of Nva-[³²P]tRNA^Leu by T252R EcLeuRS. Formation and hydrolysis of Nva-[³²P]tRNA^Leu or Leu-[³²P]tRNA^Leu by T252R LeuRS were followed in single-turnover reactions at 37 °C. Equal volumes of 30 μm T252R LeuRS with 200 μm Nva or Leu and 10 μm ATP were mixed with 2 μm [³²P]tRNA^Leu. The estimated rate constant (k_form) for Nva-[³²P]tRNA^Leu is 1.4 s⁻¹ and that for deacylation is 2.5 s⁻¹, whereas the rate constant for Leu-[³²P]tRNA^Leu formation in the same reaction conditions is 21 s⁻¹, with no hydrolysis evident within the observed time scale. The rate constants for Nva-[³²P]tRNA^Leu formation and deacylation were estimated from four independent experiments.