Fig. 4. Transient kinetic analysis of the interaction of Myosin-IE with actin and nucleotides. (A) ATP-induced dissociation of the actomyosin complex. The observed rate constants for E698 is linearly dependent on the ATP concentration in the range 5-25 µM. The apparent second-order rate constant for ATP binding to actomyosin (K₁k₊₂) was determined from the slope of the line. (B) The data over the range from 5 µM to 8 mM were fitted to a hyperbola. The rate constants for the isomerization step are given by the plateau values. (C) ADP inhibition of ATP-induced dissociation of the actomyosin complex of E698(S336E). Small amounts of ADP in the complex of E698(S336E) and pyrene-actin produced biphasic dissociation reactions. (D) Relative amplitudes of the two exponentials in dependency on the ADP concentration. The data are fitted with hyperbolae resulting in a K_AD of 12 µM for the slow phase () and the fast phase ().