FIGURE 4.
Determination of kinetic Kd (adenosine). (A) Determination of kinetic Kd (adenosine) for E. coli TrmD. Kinetic analysis was performed in single turnover conditions by rapid mixing of TrmD (0.4 μM) and adenosine (0.08–8 mM) in one syringe with tRNA (10 μM) and AdoMet (60 μM) in the second syringe (shown in the scheme). The reaction was quenched after one turnover (11 sec) by acid and the amount of product synthesis was determined. Fitting the data of concentration-dependent product synthesis to a quadratic binding equation revealed the Kd (adenosine) for TrmD. (B) Determination of kinetic Kd (adenosine) for M. jannaschii Trm5. Kinetic analysis was performed in single turnover conditions by rapid mixing of Trm5 (0.4 μM) and adenosine (0.2–17 mM) in one syringe with tRNA (16 μM) and AdoMet (60 μM) in the second syringe (shown in the scheme). The reaction was quenched after one turnover (8 sec) and the amount of product synthesis was determined. Fitting the data of the concentration-dependent product synthesis to a quadratic binding equation revealed the Kd (adenosine) for Trm5. Error bars correspond to standard deviation from three independent measurements. The inset in each graph shows the order of mixing of reagents and the reaction time. (C) A summary of Kd (AdoMet) and Kd (adenosine) for E. coli TrmD and M. jannaschii Trm5. The Kd (AdoMet) for Trm5 was previously determined (Christian et al. 2010b). The ratio of Kd (adenosine)/Kd (AdoMet) was calculated for both enzymes to reflect the competitive binding of adenosine for the AdoMet binding site. Values in parentheses correspond to the normalized ratios of Trm5 relative to TrmD. Also see Supplemental Figure S2.