Differentiating analogous tRNA methyltransferases by fragments of the methyl donor

Sinefungin inhibition of m¹G37-tRNA synthesis. (A) Analysis of initial rate of methyl transfer by TrmD (36 nM) as a function of AdoMet concentration (0–25 μM) in the presence of 20 μM E. coli tRNA^Leu and increasing concentration of sinefungin (0–4 μM). At each concentration of sinefungin, the value k_cat (in sec⁻¹) was calculated from V_max/[E], where V_max was derived from fitting the data of V₀ as a function of AdoMet concentration to the Michaelis-Menten equation, while the value of apparent K_m (in μM) was derived from the same fit. Values of k_cat and apparent K_m at each sinefungin concentration are listed. The k_cat values remained constant with increasing concentration of sinefungin, while the apparent K_m values increased; this indicates that sinefungin was a competitive inhibitor of AdoMet. (B) Analysis of initial rate of methyl transfer by Trm5 (25 nM) as a function of AdoMet concentration (0–5 μM) in the presence of 6 μM M. jannaschii tRNA^Cys and increasing concentration of sinefungin (0–60 nM). (C) The concentration-dependent apparent K_m (AdoMet) for TrmD was fit to a linear equation to derive K_m (AdoMet) from the y-intercept and K_i (sinefungin) from the x-intercept. (D) The concentration-dependent apparent K_m (AdoMet) for Trm5 was fit to a linear equation to derive K_m (AdoMet) from the y-intercept and K_i (sinefungin) from the x-intercept.