FIGURE 5.

Modulation of thiol-reactive reagent-dependent inhibition by substrates. A, inhibition of F179C activity by NEM preincubation at pH 8.5 and 5.5 in the presence of NANA (20 mm; black bars) or GlcUA (20 mm; gray bars). Dashed line indicates the level of inhibition in absence of substrate. B, inhibition of H183C activity by NEM as described in A. C, inhibition of F179C by MTEST as described in A. D, structure of NANA and GlcUA (GlcA). Control values were determined for the indicated mutants in the absence of pretreatment. For all experiments, pretreatment was for a total of 10 min: the first 8 min with substrate alone and the final 2 min with substrate and MTSET. For transport measurements the concentration of NANA was 30 nm, and the reaction was terminated at 5 min. *, p < 0.05; **, p < 0.01; ***, p < 0.0001.