Determination of enzyme kinetic constants for electrochemically mediated enzyme reactions. Application to the diaphorase–nicotinamide adenine dinucleotide system with p-methylaminophenolsulphate as an electrode carrier

Cyclic voltammetry was successfully applied to the study of the kinetics of the nicotinamide adenine dinucleotide (NADH)/diaphorase (DI)/p-methylamino-phenolsulfate (MAP) electrochemically mediated enzyme reaction. The voltammetric curves relative to the oxidation of MAP coupled with the enzymatic reaction were simulated by the DigiSim package without any simplifying assumption. The comparison between experimental and calculated curves allowed the determination of the rate constants involved in the various steps. In particular a value of 1.1×105 M−1 s−1 for the bimolecular rate constant for the reaction between enzyme and mediator and a value of 10 M−1 s−1 for the parallel competitive reaction between NADH and mediator were obtained. Other methods reported in the literature for studying the kinetics of enzymatic reactions were employed and the results were in perfect agreement with those obtained with the method based on digital simulation. A critical comparison of the merits of the different approaches is also reported.