Molecular Mechanism of ATP Hydrolysis in F₁-ATPase Revealed by Molecular Simulations and Single-Molecule Observations

Abstract

Enzymatic hydrolysis of nucleotide triphosphate (NTP) plays a pivotal role in protein functions. In spite of its biological significance, however, the chemistry of the hydrolysis catalysis remains obscure because of the complex nature of the reaction. Here we report a study of the molecular mechanism of hydrolysis of adenosine triphosphate (ATP) in F(1)-ATPase, an ATP-driven rotary motor protein. Molecular simulations predicted and single-molecule observation experiments verified that the rate-determining step (RDS) is proton transfer (PT) from the lytic water molecule, which is strongly activated by a metaphosphate generated by a preceding P(γ)-O(β) bond dissociation (POD). Catalysis of the POD that triggers the chain activation of the PT is fulfilled by hydrogen bonds between Walker motif A and an arginine finger, which commonly exist in many NTPases. The reaction mechanism unveiled here indicates that the protein can regulate the enzymatic activity for the function in both the POD and PT steps despite the fact that the RDS is the PT step.

Theory of single-molecule controlled rotation experiments, predictions, tests, and comparison with stalling experiments in F 1 -ATPase

“…The connection between binding and release events to the rotation of the γ shaft has been investigated by several groups using structural calculations (30)(31)(32)(33)(34)(35). A question arising from our study that simulations could treat is the evolution of the hydrogen-bonding network during structural displacements of a β subunit, and how it is affected by the presence of the binding nucleotide.…”

Theory for rates, equilibrium constants, and Brønsted slopes in F 1 -ATPase single molecule imaging experiments

“…As noted earlier, the present formulation of the model is not intended to apply to the ATP hydrolysis step, which is believed to involve two or more substeps, rather than just one (28,30,50). An α of 0.83 for ATP hydrolysis in tyrosyl-tRNA synthase was inferred by use of mutants, and an explanation of this high α for this reaction, where overall ΔG 0 is small, was given by Warshel, who postulated two transition states of about equal energy with a shallow minimum in between (28).…”

“…Thereby the first step had a barrier almost equal to the energy to reach the shallow well, and so α was close to unity. Electronic structure calculations in F 1 -ATPase of a more ab initio nature (30,50) indicated at least two TS's, but further studies are needed to explore these TS's, and the valley between them should be treated. Obtaining α via stalling experiments has an advantage over obtaining it via mutations because the latter may also affect λ.…”

“…Nevertheless, the general features of the model are intended to apply also to other rotation-coupled steps involving the reversible binding and release of a ligand molecule, such as ADP and P i , and to other ring-shaped NTPases, and can be tested once sufficient kinetic data become available. The model, as presently formulated, does not apply to the ATP hydrolysis (cleavage) step, because it may be a multistep process (28)(29)(30), as discussed in more detail in a later section.…”

Biological Nanomotors with a Revolution, Linear, or Rotation Motion Mechanism

“…The glutamic acid general base has been thought to activate the intervening water molecule to induce nucleophilic attack of the water molecule on the ␥ phosphate. Recent quantum chemical calculation and single-molecule analysis revealed that the role of general base may be not water activation but the enhancement of proton transfer from phosphoester to bulk solution (135). In some ATPases the glutamic acid also plays a role as a sensor for the ATP binding state and changes orientation drastically upon ATP binding (136).…”