Fig. 4.

The importance of F441 and the proposed activation mechanism are supported by in vivo functional analysis of mutants. The rate constant for electrophile-elicited K⁺ efflux was measured using a sensitive electrode-based method that allowed the initial activity to be determined accurately. (A) Electrode recordings for individual experiments in which ESG is generated by addition of NEM to cells. (B) Rate constants (mean and standard deviation) for KefC activity in the presence of the electrophile (open bars, ESG formed from N-ethylmaleimide; closed bars, SLG formed from methylglyoxal). All data represent multiple replicates, and the analytical method is described in SI Text. *No activity was observed for the F441L mutant in the presence of methylglyoxal.