Kinetic Properties of Binding of Myosin Subfragment-1 with F-Actin in the Absence of Nucleotide1

Abstract

The rate constant for the binding of myosin subfragment-1 (S-1) with F-actin in the absence of nucleotide, k₁, and that for dissociation of the F-actin-myosin subfragment-1 complex (acto-S-1), k₋₁. were measured independently. The rate of S-1 binding with F-actin was measured from the time course of the change in the light scattering intensity after mixing S-1 with various concentrations of F-actin and k₁ was found to be 2.55 × 10⁶ M⁻¹·s⁻¹ at 20°C. The dissociation rate of acto-S-1 was determined using F-actin labeled with pyrenyl iodoacetamide (Pyr-FA). Pyr-FA, with its fluorescence decreased by binding with S-1, was mixed with acto-S-1 complex and the rate of displacement of F-actin by Pyr-FA was measured from the decrease in the Pyr-FA fluorescence intensity. The k₋₁ value was calculated to be 8.5×10⁻³ s⁻¹ (or 0.51 min⁻¹). The value of the dissociation constant of S-1 from acto-S-1 complex, K_d, was calculated from K_d=k₋₁/k₁ to be 3.3×10⁻⁹ M at 20°C. K_d was also measured at various temperatures (0–30°C), and the thermodynamic parameters, ΔG°, ΔH°, and ΔS° were estimated from the temperature dependence of K_d to be —11.3 kcal/mol, + 2.5 kcal/mol, and +47 cal/deg.mol, respectively. Thus, the binding of the myosin head with F-actin was shown to be endothermic and entropy-driven.