Association Kinetics and Binding Constants of Nucleoside Triphosphates with G‐Actin

Abstract

The dissociation of the complex between 1:N⁶‐ethenoadenosine 5′‐triphosphate (ɛP) and G‐actin was initiated by dilution to concentrations between 1 μM and 5 nM and monitored by the fluorescence change of ɛP. The results were quantitatively explained by a two‐step mechanism: a reversible dissociation of the actin · nucleotide complex followed by a fast irreversible inactivation of nucleotide‐free G‐actin. Under normal conditions (0.8 mM CaCl₂, pH 8.2, 21 °C), the rate‐limiting step was the dissociation of the nucleotide· G‐actin complex. The half‐time of the dissociation of ɛP from G‐actin was 290 s as compared to only 13 s for the following denaturation step of nucleotide‐free actin. 1 mM EDTA highly accelerated the dissociation step and, regardless of its concentration, the complex dissociated quantitatively within 1 min. Addition of Ca²⁺ within 20 s after EDTA addition induced a re‐association of ɛP with nucleotide‐free but still native G‐actin. This reversal was kinetically resolved by means of a multimixing stopped‐flow apparatus. The association rate constant was 6 × 10⁶ M⁻¹ s⁻¹. From the association and dissociation rate constant, a value of 2.5 × 10⁹ M⁻¹ was calculated for the binding constant of ɛP to G‐actin. The binding constant of ATP (1.4 × 10¹⁰ M⁻¹) was derived from the relative binding constant of ɛP and ATP as determined by fluorescence titration of ɛP · G‐actin with ATP. These binding constants are 10³–10⁴ times higher than values reported earlier on the basis of more indirect data.