Catalysis by ATP synthase: mechanistic, kinetic and thermodynamic characteristics

Mechanistic, kinetic and thermodynamic aspects of ATP catalysis by ATP synthase have been determined and analyzed. Reversibility and irreversibility of catalysis in ATP synthase represent two contrasting modes of catalysis with important implications for the molecular mechanism of ATP synthesis. To shed light on these aspects, we have developed kinetic schemes for ATP synthesis and hydrolysis; analysis of these schemes reveals several novel features and provides new directions for further research. First, the ratio of bound 32Pi to total bound 32P can be expressed in terms of the rate constants of the elementary catalytic steps, which are characteristic properties of the system; therefore, results of classical cold chase/acid quench 32Pi experiments interpreted in terms of an equilibrium distribution of bound substrate and product at the catalytic site can be explained by an irreversible mode of catalysis. Second, characterization of the mechanistic and kinetic properties reveals the absence of cooperativity in ATP synthase, and that product release precedes substrate binding. Third, ΔpH and Δψ are kinetically inequivalent in driving ATP synthesis, and ΔμH is not the true driving force for ATP synthesis. Thermodynamic analysis of ATP synthesis reveals a dynamically electrogenic but overall electroneutral mode of ion transport across the membrane. The P/O ratio based on the torsional mechanism was obtained and was shown to explain the experimental observations of the past 50 years and to be in agreement with the thermodynamic calculations. Taken together, these findings necessitate a paradigm shift for understanding the molecular mechanism of ATP synthesis.