Figure 5.

Determination of carbachol binding constants with carbamoylated AChE from the fluorescence of AChE-bound thioflavin T after the carbachol reactions have reached a final steady state. F_ECN values from mixtures of AChE (70 - 155 nM), thioflavin T (1.0 - 15.6 μM, as noted), and the indicated concentration of carbachol were determined from stopped-flow traces as in Figure 3. The F_ECN and then F_ECN/f_ECL data were analyzed with eq 11 as described for F_EM and then F_EM/f_EL in Figure 4. Parameters obtained from the data fitting are shown in Table 2. The dotted lines show the fit when carbachol is assumed to bind to only one site, (e.g., carbachol and thioflavin T compete for the P-site: (i_ECK_ECS)⁻¹ and i_EC2⁻¹ fixed at 0 with fitted K_M+S = 57).