Beyond binding change: the molecular mechanism of ATP hydrolysis by F1-ATPase and its biochemical consequences

F1-ATPase is a universal multisubunit enzyme and the smallest-known motor that, fueled by the process of ATP hydrolysis, rotates in 120o steps. A central question is how the elementary chemical steps occurring in the three catalytic sites are coupled to the mechanical rotation. Here, we performed cold chase promotion experiments and measured the rates and extents of hydrolysis of preloaded bound ATP and promoter ATP bound in the catalytic sites. We found that rotation was caused by the electrostatic free energy change associated with the ATP cleavage reaction followed by Pi release. The combination of these two processes occurs sequentially in two different catalytic sites on the enzyme, thereby driving the two rotational sub-steps of the 120o rotation. The mechanistic implications of this finding are discussed based on the overall energy balance of the system. General principles of free energy transduction are formulated, and their important physical and biochemical consequences are analyzed. In particular, how exactly ATP performs useful external work in biomolecular systems is discussed. A molecular mechanism of steady-state, trisite ATP hydrolysis by F1-ATPase, consistent with physical laws and principles and the consolidated body of available biochemical information, is developed. Taken together with previous results, this mechanism essentially completes the coupling scheme. Discrete snapshots seen in high-resolution X-ray structures are assigned to specific intermediate stages in the 120o hydrolysis cycle, and reasons for the necessity of these conformations are readily understood. The major roles played by the '' minor '' subunits of ATP synthase in enabling physiological energy coupling and catalysis, first predicted by Nath ' s torsional mechanism of energy transduction and ATP synthesis 25 years ago, are now revealed with great clarity. The working of nine-stepped (bMF1, hMF1), six-stepped (TF1, EF 1), and three-stepped (PdF1) F1 motors and of the a3 ss 3. subcomplex of F1 is explained by the same unified mechanism without invoking additional assumptions or postulating different mechanochemical coupling schemes. Some novel predictions of the unified theory on the mode of action of F1 inhibitors, such as sodium azide, of great pharmaceutical importance, and on more exotic artificial or hybrid/chimera F1 motors have been made and analyzed mathematically. The detailed ATP hydrolysis cycle for the enzyme as a whole is shown to provide a biochemical basis for a theory of '' unisite '' and steady-state multisite catalysis by F1-ATPase that had remained elusive for a very long time. The theory is supported by a probabilitybased calculation of enzyme species distributions and analysis of catalytic site occupancies by Mg-nucleotides and the activity of F1-ATPase. A new concept of energy coupling in ATP synthesis/hydrolysis based on fundamental ligand substitution chemistry has been advanced, which offers a deeper understanding, elucidates enzyme activation and catalysis in a better way, and provides a unified molecular explanation of elementary chemical events occurring at enzyme catalytic sites. As such, these developments take us beyond binding change mechanisms of ATP synthesis/hydrolysis proposed for oxidative phosphorylation and photophosphorylation in bioenergetics.

Automated summary

F1-ATPase is a small motor that rotates in 120o steps fueled by ATP hydrolysis. This study reveals that the rotation is driven by the electrostatic free energy change associated with ATP cleavage reaction and Pi release. This finding has important implications for understanding the mechanical working mechanism of F1-ATPase and the transduction of free energy in biomolecular systems.