Determination of the six rate constants of a three-state enzymatic network and a noninvasive test of detailed balance.

The Michaelis-Menten mechanism is unanimously recognized by experimentalists and theoreticians as the reference model for the description of enzymatic catalysis. The recent explosion in the diversity of fluorescent probes solves the problem of in situ observation of proteins and the experimental investigation of enzymatic dynamics, which determines the Michaelis constant or a small number of relaxation times, is becoming more and more common. We propose a protocol for the full characterization of enzyme kinetics in the framework of the Michaelis-Menten mechanism. The method relies on the measurement of the oscillation amplitude of the enzymatic concentrations, when the biological medium is submitted to a temperature modulation of a few degrees. Analytical expressions of all the rate constants as functions of the concentration amplitudes are derived. The noninvasive character of the perturbation and the assessable uncertainty on the rate constant values make an in situ test of detailed balance possible.