Quantifying the flux as the driving force for nonequilibrium dynamics and thermodynamics in non-Michaelis-Menten enzyme kinetics.

The driving force for active physical and biological systems is determined by both the underlying landscape and nonequilibrium curl flux. While landscape can be experimentally quantified from the histograms of the collected real-time trajectories of the observables, quantifying the experimental flux remains challenging. In this work, we studied the single-molecule enzyme dynamics of horseradish peroxidase with dihydrorhodamine 123 and hydrogen peroxide (H2O2) as substrates. Surprisingly, significant deviations in the kinetics from the conventional Michaelis-Menten reaction rate were observed. Instead of a linear relationship between the inverse of the enzyme kinetic rate and the inverse of substrate concentration, a nonlinear relationship between the two emerged. We identified nonequilibrium flux as the origin of such non-Michaelis-Menten enzyme rate behavior. Furthermore, we quantified the nonequilibrium flux from experimentally obtained fluorescence correlation spectroscopy data and showed this flux to led to the deviations from the Michaelis-Menten kinetics. We also identified and quantified the nonequilibrium thermodynamic driving forces as the chemical potential and entropy production for such non-Michaelis-Menten kinetics. Moreover, through isothermal titration calorimetry measurements, we identified and quantified the origin of both nonequilibrium dynamic and thermodynamic driving forces as the heat absorbed (energy input) into the enzyme reaction system. Furthermore, we showed that the nonequilibrium driving forces led to time irreversibility through the difference between the forward and backward directions in time and high-order correlations were associated with the deviations from Michaelis-Menten kinetics. This study provided a general framework for experimentally quantifying the dynamic and thermodynamic driving forces for nonequilibrium systems.