Rate of hydrolysis in ATP synthase is fine-tuned by α-subunit motif controlling active site conformation

Abstract

Computer-designed artificial enzymes will require precise understanding of how conformation of active sites may control barrier heights of key transition states, including dependence on structure and dynamics at larger molecular scale. F o F 1 ATP synthase is interesting as a model system: a delicate molecular machine synthesizing or hydrolyzing ATP using a rotary motor. Isolated F 1 performs hydrolysis with a rate very sensitive to ATP concentration. Experimental and theoretical results show that, at low ATP concentrations, ATP is slowly hydrolyzed in the so-called tight binding site, whereas at higher concentrations, the binding of additional ATP molecules induces rotation of the central γ-subunit, thereby forcing the site to transform through subtle conformational changes into a loose binding site in which hydrolysis occurs faster. How the 1-Å-scale rearrangements are controlled is not yet fully understood. By a combination of theoretical approaches, we address how large macromolecular rearrangements may manipulate the active site and how the reaction rate changes with active site conformation. Simulations reveal that, in response to γ-subunit position, the active site conformation is fine-tuned mainly by small α-subunit changes. Quantum mechanics-based results confirm that the sub-Ångström gradual changes between tight and loose binding site structures dramatically alter the hydrolysis rate.enzyme catalysis | molecular dynamics | protein structure M ost cellular processes depend on ATP as a free energy source. Among a wide spectrum of organisms, the key enzyme responsible for maintaining the ATP concentration is F o F 1 ATP synthase, with synthesis being driven by a transmembrane electrochemical gradient (1). The chemical reaction takes place in the F 1 domain at active sites located in the three αβ-subunit pairs surrounding a central α-helical coiled coil, the γ-subunit. It has been shown in single-molecule experiments on F 1 that ATP synthesis and ATP hydrolysis are coupled to γ-subunit rotations of opposite directions (1, 2). Early kinetic studies revealed a strong ATP concentration dependence for the hydrolysis rate that was explained involving both unisite and multisite catalysis: low ATP concentrations, with only one active site populated by unisite catalysis (3, 4), whereas at physiological (high) ATP concentrations, multisite catalysis takes over, resulting in several orders of magnitude increase in hydrolysis rate (5). This remarkable catalytic cooperativity of the enzyme could be an effect of the mechanochemical coupling between the active sites. In crystal structures, three conformations of the catalytically active sites can be seen, assigned as tight binding site (α TP /β TP ; T), loose binding site (α DP /β DP ; D), and empty site (α E / β E ; E) (6-10). During ATP hydrolysis, each α/β-subunit pair changes conformation along the cycle: E→T→D→E, each step associated with a 120°rotation of the γ-subunit (5, 7, 10, 11). Single-molecule studies supported by simulations indicate that the 120°rotation...

Torque, chemistry and efficiency in molecular motors: a study of the rotary–chemical coupling in F1-ATPase

“…2013 ). The conformational dependence of the catalytic step has been explored recently by Beke-Somfai et al . (2013) , who used QM/MM calculations and phenomenological description to model the process.…”

“…The conformational dependence of the catalytic step has been explored recently by Beke-Somfai et al . (2013), who used QM/MM calculations and phenomenological description to model the process. It was concluded that the activation barrier is reduced significantly upon T to D change of the active site.…”

Phosphate release coupled to rotary motion of F 1 -ATPase

“…These simulations also allow us to explore the coupling between the 40°s ubstep and P i release, and to examine the underlying molecular mechanisms. F 1 has been studied by molecular simulations at various levels of resolution, including quantum chemical calculations of ATP hydrolysis (25)(26)(27), all-atom simulations of conformational changes in the β-subunit (28,29), of ATP release (30), of fluctuations in the complex (31,32), and of γ-rotation (33,34), as well as coarse-grained simulations of γ-rotation (35)(36)(37). As in experiment and in earlier simulations (33), we apply external torque to modulate the rates of the functional processes, including P i release.…”

ATP Hydrolysis in the RecA–DNA Filament Promotes Structural Changes at the Protein–DNA Interface

“…17,18 Here the O W ···γP distance is 2.10 Å, while the γP···O(-βP) distance is 2.71 Å, which is somewhat longer than those of observed for the other enzymes.…”