Fig. 3.

Detection and quantitation of covalent intermediates in the reactions of AHAS II mutant Arg276Lys. Steady-state intermediate distribution of AHAS II mutant Arg276Lys in the presence of 100 mM pyruvate (A) or 50 mM pyruvate and 50 mM 2-ketobutyrate (B) in 0.1 M KPi at 37°C. Reactions were quenched after 300 msec, and the protein-free solutions obtained after centrifugation were analyzed by ¹H NMR spectroscopy. Note that the fingerprint region (7.0–8.0 ppm) is zoomed 3- and 2-fold in A and B, respectively, relative to the downfield region where the ThDP C2-H appears.