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

Abstract

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 timeindependent 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 reaction. 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.

Square‐wave Voltammetry of Two‐step Surface Electrode Mechanisms Coupled with Chemical Reactions – A Theoretical Overview

“…This is achievable, since all of the mechanisms (2), (3) and (4) have quite specific SW voltammetric patterns, regardless whether both steps occur at quite different or at very same potential. When both electrode steps of a given two‐step surface mechanism are separated for 300 mV or even more, then we can explore the methods elaborated in to get insight to all relevant kinetic and thermodynamic parameters. The method of “quasireversible maximum” can be explored to get insight into kinetics of electrode reactions in case of two well‐separated SW peaks at all elaborated two‐step mechanisms, except at the surface EECrev mechanism featuring high values of equilibrium constant of follow up chemical step .…”

“…In the last two decades, this methodology is successfully explored to quantify important compounds and to determine kinetic and thermodynamic parameters relevant to relevant lipophilic biological systems that undergo successive multi‐step redox transformation . Moreover, our group and others presented several theoretical models under voltammetric conditions that provide a basis for designing simple methods to recognize a particular electrode mechanism. In addition, plenty of simple methods are provided in our theoretical works allowing elegant access to kinetic and thermodynamic parameters relevant to many lipophilic redox active systems.…”

Theoretical Analysis of a Surface Catalytic Mechanism Associated with Reversible Chemical Reaction Under Conditions of Cyclic Staircase Voltammetry

“…Indeed, a good theoretical basis is needed to understand many phenomena when given redox enzymes or surface-active redox compounds are voltammetrically analyzed [4,5]. Among the surface redox systems studied with voltammetric techniques, those coupled with chemical reactions are of outmost importance [1,4,[9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. This is because voltammetric studies on such systems can provide valuable information about their chemical features [1,[4][5][6][7].…”

“…This is because voltammetric studies on such systems can provide valuable information about their chemical features [1,[4][5][6][7]. In all surface mechanisms complicated with chemical reactions, the surface catalytic (regenerative) mechanism EC' (or, more precisely, the surface EC irr ' mechanism, where "irr" stands for irreversible) is most comprehensively studied under voltammetric conditions [1][2][3][4][22][23][24][25][26][27]. Voltammetric theories developed on this mechanism provide simple approaches to get access into kinetics and thermodynamics of electron transfer and the chemical reactivity of many compounds [4,[25][26][27].…”

Protein‐film Voltammetry of Two‐step Electrode Enzymatic Reactions Coupled with an Irreversible Chemical Reaction of a Final Product – A Theoretical Study in Square‐wave Voltammetry

“…This holds true especially for the theory of two-step electrode reactions of many redox enzymes under conditions of pulse voltammetric techniques. Although several theoretical models have been reported in the past decade related to the features of two-step electrode reactions under conditions of cyclic staircase voltammetry [1,[7][8][9][10][11][12] and in square-wave voltammetry for systems with one and two electrode steps [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30], yet there are still many new aspects emerging that need additional considerations. In this work we present results of a new theoretical model in square-wave voltammetry (SWV).…”