Time-Independent Methodology to Access Michaelis-Menten Constant by Exploring Electrochemical-Catalytic Mechanism in Protein-Film Cyclic Staircase Voltammetry

: Protein-film voltammetry is recognized as a very efficient tool in mechanistic enzymology, but it is also seen as a relevant approach to gain thermodynamic and kinetic information related to the redox chemistry of many enzymes. This technique requires a small amount of redox enzyme, whose molecules form monomolecular film on the working electrode surface. In this paper we present a simple and time - independent cyclo-voltammetric method for the determination of kinetics of the chemical step of an electrochemical-catalytic (EC’) mechanism in protein- film scenario. Theoretical results of a surface EC’ mechanism show that the limiting cyclo -voltammetric catalytic current, measured at large overpotentials, depends solely on the rate of the chemical regenerative re action. At large overpotentials, the limiting current of the steady-state cyclic voltammograms is independent on all kinetics and thermodynamic parameters related to the electrode reaction of adsorbed enzyme. The approach proposed relies on the dependence of the magnitude of limiting current of the experimental cyclic steady-state voltammograms as a function of the substrate concentration.