Electrocatalytic Oxidation of Dihydronicotinamide Adenine Dinucleotide with Ferrocene Carboxylic Acid by Diaphorase fromClostridium kluveri. Remarks on the Kinetic Approaches Usually Adopted

Abstract

The kinetics of the electron transfer between NADH and ferrocenecarboxylic acid (FMCA) catalyzed by diaphorase from Clostridium kluveri (EC 1.8.1.4.) was studied by voltammetry. A commercially available digital simulation program permitted one to determine the rate constants of the catalytic system by a ®tting procedure. A rate constant of 3610 4 M À1 s À1 was found for the reaction between diaphorase and mediator while a value of 8 M À1 s À1 was found for the parallel competitive reaction between NADH and mediator. In addition a comparison of the present method to other data treatment procedures reported in the literature and a brief evaluation of their limits of applicability were carried out.

A General Method for the Electrochemical Evaluation of the Bimolecular Rate Constant in Enzyme Catalyzed Reaction Kinetics

“…The insertion of the extrapolated ratios I lim /I d for the FMCA and MAP mediation in the working curve in Figure 1 yields the enzymatic parameters k 3.1610 4 M 71 s 71 and k 1.1610 5 M 71 s 71 , respectively. These values are in good agreement with those obtained by a curve ®tting procedure performed via digital simulation [5,7].…”

“…This equation shows the dependence of the ratios I lim /I d on the scan rate v, on the number n of electrons exchanged by the mediator and on the parameter x which is equal to 2 for a 2:1 stoichiometry of the reaction between mediator and enzyme. The introduction of the parameter x allows one to determine directly the correct k value which otherwise resulted to be twice the right value [4,5]. Figure 1 shows the plot of 0.1992 (I lim /I d ) 2 (nFv/xRT) versus k[E] (solid line) which is a straight line with slope equal to 1.…”

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.…”

“…The two-electron electrode reactions of VK 3 and of ARS were simulated with differences in the potentials (D E8' E8' 2 À E8' 1 ) between the first and the second step of electron transfer process of about 180 mV in order to obtain the observed wave of two merged one-electron transfers [25]. The diffusion coefficients for NADH/NAD and DI were taken from the literature [18] and the unavailable diffusion coefficients and the heterogeneous electron transfer reaction constants of both one-electron steps of ARS and VK 3 were chosen to match the experimental curves. The voltammetric behaviors appear as close to reversible owing to the chosen heterogeneous constants (k s,1 0.01 cm s À 1 ; k s,2 0.006 cm s À 1 ; a 1 a 2 0.5 for the mediator VK 3 ; k s,1 0.01 cm s À 1 ; k s,2 0.01 cm s À 1 ; a 1 a 2 0.5 for the mediator ARS) and the very slow scan rate used (v 5 mV s À 1 ).…”

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

“…The evaluation of the kinetic constant is obtained by the use of appropriate working curves [6,8]. Very recently a commercially available software package, DigiSim [9], has been successfully applied to the problem at hand [10,11]. Unlike the statement of Yokoyama et al [8] the use of the DigiSim software is very convenient to simulate electrochemically drawn enzyme reactions since it permits one to handle several chemical reactions coupled to the charge transfer step and to simulate steady state conditions with a suitable choice of the kinetic constants and of the concentrations of the reactants.…”