Physical Confinement Modulates the Rate-Limiting Transition in the Release of Phosphate from Actin Filaments

The nucleotide state and rates of transitions between states regulate the dynamics of ATPases. Slow inorganic phosphate (P i ) release following ATP hydrolysis is often rate-limiting and associated with key conformational changes. Actin filaments offer a unique opportunity to understand the fundamentals of phosphate release, because identical subunits at filament ends and the interior release P i at markedly different rates. The molecular origin of this difference is debated, so we employed extensive all-atom molecular dynamics simulations to characterize P i release from different subunits within an actin filament. The dissociation rates of P i from ADP-Mg 2+ in the active site correlate with biochemically measured P i release rates and scale inversely with the numbers of water molecules in the cavity surrounding the γ-phosphate. Simulations show that egress of P i through protein channels, including through the N111-R177 backdoor, is not rate-limiting and, importantly, that subunits at the filament ends use alternative egress pathways.