Determination of Enzyme Kinetic Constants for Electrochemically Mediated Enzyme Reactions. Application to the Diaphorase-Nicotinamide Adenine Dinucleotide System withp-Methylaminophenolsulfate as an Electron Carrier

Abstract

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.

EC mechanism of an adsorbed redox couple. Volume vs surface chemical reaction

“…In the latter paper Laviron initiated a remarkable discussion on the question: Can voltammetry distinguish the volume versus surface following chemical reactions? The question is of particular importance for contemporary electroanalytical methods dealing with physiologically active compounds [20][21][22][23][24][25] and catalytic systems [26], whose electrode reactions involve adsorption and follow-up chemical reactions. Unfortunately, LavironÕs theory revealed that these two types of chemical reactions cannot be discriminated by means of an electrochemical method only.…”

Electrocatalytic Oxidation of NADH by Oxidized s‐Adenosyl‐L‐Methionine (SAMe): Application to NADH and SAMe Determinations

“…The intrinsic drawbacks of the electrochemical detection of NADH on solid electrodes through its oxidation process, i.e., large overpotentials and fouling of the electrode surface due to the accumulation of high molecular weight oxidation products, can be overcome by means of adequate pretreatments of the electrode surface [1], developing new electrode materials [2 ± 4], enzymatic oxidation [5,6] or using electron-transfer mediators. The latter is the most widely extended strategy.…”

Kinetic and Thermodynamic Aspects of NAD-Related Enzyme-Linked Mediated Bioelectrocatalysis

“…To confirm that the overall equilibrium constant is not the sole parameter determining the characteristics of the bioelectrocatalysis we considered the mediator FMCA in the place of MAP (E8' FMCA 0.440 V at pH 7.0 [18]; E8' MAP 0.288 V at pH 7.0 [19]), that implies an overall electron transfer reaction from the mediator to L-lactate with K eq 2 Â 10 21 instead of K eq 1.4 Â 10 16 . Figure 5 shows the catalytic waves relative to the DI-LDH linked system mediated by FMCA.…”