TABLE 1

K_m and V_max values of EF, EF3, and various EF3 mutants

AC toxin activities were determined as described under Materials and Methods. K_m and V_max values were obtained by nonlinear regression analysis of substrate-saturation experiments and are the means ± S.D. of at least three independent experiments performed in triplicates. For the determination of the V_max and K_m of EF, EF3, and EF3(F586A), reaction mixtures contained 100 μM free Ca²⁺, 5 mM free Mn²⁺, 100 μM EGTA, 0.2-1.0 μCi/tube [α-³²P]ATP, 100 μM cAMP, 100 nM calmodulin, and 10 pM enzyme in 75 mM Tris/HCl, pH 7.4. In the case of the other EF3 mutants, reaction mixtures contained 0.4 μCi/tube [α-³²P]ATP and 2 nM enzyme. For determination of V_max and K_m without CaM and Ca²⁺, the enzyme concentration was 5 nM. The unlabeled ATP/Mn²⁺ concentrations ranged from 100 nM to 4 mM as appropriate to obtain saturated curves.