Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments

Abstract

Reliable estimation of the kinetic parameters of redox enzymes by taking into account mass transport towards rotating electrodes in protein film voltammetry experiments. Electrochimica Acta, Elsevier, 2017Elsevier, , 245, pp.1059Elsevier, -1064Elsevier, . 10.1016Elsevier, /j.electacta.2017 AbstractIn Protein Film Voltammetry, a redox enzyme is immobilized on a rotating electrode in a configuration allowing fast, direct electron transfer. This technique is used to probe the mechanism of enzymes by quantitatively interpreting the response in current as a function of the experimental conditions. Limitation by mass transport of the substrate towards the electrode may obscure important features and complicate the analysis of the enzymatic response, so much so that the enzyme has high activity. In this work, we derive equations taking into account mass transport of substrate, for the steady-state current generated by an enzyme following Michaelis-Menten kinetics and immobilized onto a hydrodynamic (e.g. rotating) electrode. We use these equations to model the current response of films of CO-dehydrogenase, a metalloenzyme that catalyzes the oxidation of CO, to transient exposures to its gaseous substrate. We show that neglecting transport yields poor fits and overestimated, unreliable, values of K m (even when using the Koutecky-Levich approximation), whereas taking into account transport yields much better fits and more reliable parameters. We reinterpret previously published data by taking into account transport limitations.

The two CO-dehydrogenases of Thermococcus sp. AM4

“…The exchange of gas between the electrochemical cell and the atmosphere of the glove box results in an exponential decrease in the concentration of CO in the cell [21]. We have quantitatively modeled the electrochemical signal by taking into account mass-transport limitations, using the software QSoas [22] (http://qsoas.org) and the method described in [23].…”

“…To characterize the reactivity of the enzymes with O 2 , we have used the method described in ref. [23]: the experiments were performed in a pH 7 mixed buffer through chronoamperometry at -0.31 V vs. SHE, by injecting in the cell six consecutive aliquots of 100 µL CO-saturated buffer. Injections 1, 3 and 5 were preceded by 20 s poise at -0.76 V, and an aliquot of O 2 -saturated (or air-saturated) buffer was injected 10 s after injection 3 (see Supplementary Figure S7).…”

CODH‐IV: A High‐Efficiency CO‐Scavenging CO Dehydrogenase with Resistance to O2

“…Fitting the model in Ref. [12] to the data (dotted lines in Figure 1) yielded K m values of 8 AE 1 mm (CODH-II) and 47 AE 3nm (CODH-IV) (at 25 8 8C). Figure 2s hows the values of K m (determined using electrochemical experiments), k cat (determined using solution assays) and k eff = k cat /K m for temperatures ranging from 25 8 8C to 60 8 8C.…”

CODH‐IV: eine hocheffiziente CO‐Dehydrogenase mit Resistenz gegen O2

“…Die Anpassung des Modells aus Lit. an die Daten (gestrichelte Linien in Abbildung ) ergab K m ‐Werte von 8±1 μ m (CODH‐II) und 47±3 n m (CODH‐IV) (bei 25 °C).…”

“…(9) aus Lit. (gestrichelte Linien). Um den Verglich zu erleichtern, wurde die Zeit anhand der Zeitkonstante τ CO (exponentielle Abnahme der CO‐Konzentration) normalisiert.…”