Changes in the adenine nucleotide and inorganic phosphate content of Escherichia coli F1-ATPase during ATP synthesis in dimethyl sulphoxide.
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ABSTRACT:
SUBMITTER: Beharry S
PROVIDER: S-EPMC1132940 | biostudies-other | 1992 Sep
REPOSITORIES: biostudies-other
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Project description:Most of the cellular ATP in living organisms is synthesized by the enzyme F(1)F(o)-ATP synthase. The water soluble F(1) part of the enzyme can also work in reverse and utilize the chemical energy released during ATP hydrolysis to generate mechanical motion. Despite the availability of a large amount of biochemical data and several x-ray crystallographic structures of F(1), there still remains a considerable lack of understanding as to how this protein efficiently converts the chemical energy released during the reaction ATP + H(2)O --> ADP + P(i) into mechanical motion of the stalk. We report here an ab initio QM/MM study of ATP hydrolysis in the beta(TP) catalytic site of F(1). Our simulations provide an atomic level description of the reaction path, its energetics, and the interaction of the nucleotide with the protein environment during catalysis. The simulations suggest that the reaction path with the lowest potential energy barrier proceeds via nucleophilic attack on the gamma-phosphate involving two water molecules. Furthermore, the ATP hydrolysis reaction in beta(TP) is found to be endothermic, demonstrating that the catalytic site is able to support the synthesis of ATP and does not promote ATP hydrolysis in the particular conformation studied.
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Project description:F1-ATPase, the catalytic domain of ATP synthase, synthesizes most of the ATP in living organisms. Running in reverse powered by ATP hydrolysis, this hexameric ring-shaped molecular motor formed by three ??-dimers creates torque on its central ?-subunit. This reverse operation enables detailed explorations of the mechanochemical coupling mechanisms in experiment and simulation. Here, we use molecular dynamics simulations to construct a first atomistic conformation of the intermediate state following the 40° substep of rotary motion, and to study the timing and molecular mechanism of inorganic phosphate (Pi) release coupled to the rotation. In response to torque-driven rotation of the ?-subunit in the hydrolysis direction, the nucleotide-free ??E interface forming the "empty" E site loosens and singly charged Pi readily escapes to the P loop. By contrast, the interface stays closed with doubly charged Pi. The ?-rotation tightens the ATP-bound ??TP interface, as required for hydrolysis. The calculated rate for the outward release of doubly charged Pi from the ??E interface 120° after ATP hydrolysis closely matches the ~1-ms functional timescale. Conversely, Pi release from the ADP-bound ??DP interface postulated in earlier models would occur through a kinetically infeasible inward-directed pathway. Our simulations help reconcile conflicting interpretations of single-molecule experiments and crystallographic studies by clarifying the timing of Pi exit, its pathway and kinetics, associated changes in Pi protonation, and changes of the F1-ATPase structure in the 40° substep. Important elements of the molecular mechanism of Pi release emerging from our simulations appear to be conserved in myosin despite the different functional motions.
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Project description:The rotation of the central stalk of F1-ATPase is driven by energy derived from the sequential binding of an ATP molecule to its three catalytic sites and the release of the products of hydrolysis. In human F1-ATPase, each 360° rotation consists of three 120° steps composed of substeps of about 65°, 25°, and 30°, with intervening ATP binding, phosphate release, and catalytic dwells, respectively. The F1-ATPase inhibitor protein, IF1, halts the rotary cycle at the catalytic dwell. The human and bovine enzymes are essentially identical, and the structure of bovine F1-ATPase inhibited by IF1 represents the catalytic dwell state. Another structure, described here, of bovine F1-ATPase inhibited by an ATP analog and the phosphate analog, thiophosphate, represents the phosphate binding dwell. Thiophosphate is bound to a site in the ?(E)?(E)-catalytic interface, whereas in F1-ATPase inhibited with IF1, the equivalent site is changed subtly and the enzyme is incapable of binding thiophosphate. These two structures provide a molecular mechanism of how phosphate release generates a rotary substep as follows. In the active enzyme, phosphate release from the ?(E)-subunit is accompanied by a rearrangement of the structure of its binding site that prevents released phosphate from rebinding. The associated extrusion of a loop in the ?(E)-subunit disrupts interactions in the ?(E)?(E-)catalytic interface and opens it to its fullest extent. Other rearrangements disrupt interactions between the ?-subunit and the C-terminal domain of the ?(E)-subunit. To restore most of these interactions, and to make compensatory new ones, the ?-subunit rotates through 25°-30°.
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