Enzymatic mechanisms and electron transfer mediation in chronoamperometric biosensors

Abstract

Electron transfer mediation to an anode, whose potential is judiciously controlled, provides the conceptual basis for the development of novel chronoamperometric biosensors. The mediators are appropriately selected redox couples (Ox/Red) which are amenable to recycling in the following type of enzyme E catalyzed reaction sequence

$$\begin{gathered} {\text{Substrate + Ox}}{}^{\text{E}} \to {\text{ Product + Red }} \hfill \\ {\text{Red }} \to {\text{ Ox + }}ne^ - {\text{ }} \hfill \\ \end{gathered}$$

Glucose Oxidase (GOD) and Alcohol Oxidase (AOD) exhibit remarkably different pattern of behavior. While GOD is highly specific for the substrate, β-D-glucose, this enzyme can accommodate a variety of oxidants as co-substrates. On the other hand, AOD exhibits broader substrate specificity (short-chain primary alcohols) but accepts only one known oxidant as co-substrate, viz. dioxygen. This paradox is accounted for by the fact that GOD is known to function via a so-called ping-pong mechanism of enzymatic catalysis, which involves successive binding on two different sites, viz.

• (a) a ‘substrate site’ that is capable of binding exclusively β-D-glucose

• (b) a relatively non-selective ‘co-substrate site’ for the oxidant.

In contradistinction, AOD's catalytic mechanism implicates a ternary intermediate, where both the substrate and the co-substrate are bound to the same site of the enzyme. Accordingly, we postulate that AOD is a ‘true oxidase’, which is selective to dioxygen as the sole acceptable cosubstrate, while GOD is an example of a ‘pseudo-oxidase’ which can accept several co-substrates on a less selective site.