Mechanical modulation of catalytic power on F1-ATPase

Abstract

The conformational fluctuation of enzymes has a crucial role in reaction acceleration. However, the contribution to catalysis enhancement of individual substates with conformations far from the average conformation remains unclear. We studied the catalytic power of the rotary molecular motor F(1)-ATPase from thermophilic Bacillus PS3 as it was stalled in transient conformations far from a stable pausing angle. The rate constants of ATP binding and hydrolysis were determined as functions of the rotary angle. Both rates exponentially increase with rotation, revealing the molecular basis of positive cooperativity among three catalytic sites: elementary reaction steps are accelerated via the mechanical rotation driven by other reactions on neighboring catalytic sites. The rate enhancement induced by ATP binding upon rotation was greater than that brought about by hydrolysis, suggesting that the ATP binding step contributes more to torque generation than does the hydrolysis step. Additionally, 9% of the ATP-driven rotary step was supported by thermal diffusion, suggesting that acceleration of the ATP docking process occurs via thermally agitated conformational fluctuations.

Operation mechanism of FoF1‐adenosine triphosphate synthase revealed by its structure and dynamics

“…The stator a 3 b 3 ring has the intrinsic cooperativity necessary to achieve rotary catalysis among the three catalytic sites. However, this result is not inconsistent with the observations that the rates and equilibriums of the F 1 reactions are controlled by the rotary angle of the c-subunit (14,18,(25)(26)(27). Without the c-subunit, the rate and efficiency of the unidirectional rotary catalysis was distinctly lower than that for F 1 .…”

“…Thus, it was proposed that interactions with the c-subunit control the conformational and catalytic states of each b-subunit to sequentially generate torque (24). The single-molecule manipulation experiments showed that by controlling the rotary angle of the c-subunit alone, the rate and equilibrium of the ATP hydrolysis/synthesis reaction on F 1 can be modulated and even reversed, and reinforced the contention (14,18,(25)(26)(27). These results caused the elevation of the c-subunit to the position of a ''dictator,'' completely controlling the reaction catalyzed by F 1 .…”

What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

“…The linearity condition was not fulfilled for the ATP binding step but was roughly fulfilled for the hydrolysis although the present data are relatively sparse (Fig. 3c in Watanabe et al 2012).…”

“…A particular example would be to use a collision theory picture (Volkán-Kacsó & Marcus, 2015) of ATP binding to the weak binding site of an empty β subunit in which the pre-exponential term A is a collision frequency Z, and a term related to an ATP binding outside of the β subunit. For the binding and release of ATP there are extensive data obtained by different methods (Adachi et al 2012;Braig et al 2000;Spetzler et al 2009;Watanabe et al 2010Watanabe et al , 2012Yasuda et al 2001). In the ATP binding an ATP first forms a collision complex (Oster & Wang, 2000) with the F 1 -ATPase, followed by an ATP binding as the next step (an 80°step) in the overall hydrolysis.…”

“…Boyer, 1993 and references therein). To this end we describe a chemical-mechanical theory (Volkán-Kacsó & Marcus, 2015) that we have formulated to treat several types of singlemolecule experiments on this biomolecular motor (Adachi et al 2012;Watanabe et al 2012). The theory draws heavily on a theory of electron and other transfers in solution (Marcus, 1968(Marcus, , 1993Marcus & Sutin, 1985).…”