Non-enzymatic interaction of reaction products and substrates in peroxidase catalysis

A quantitative approach for estimation of the non-enzymatic interaction between ammonium 2,2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation product and a poorly oxidized substrate was developed using a system including tobacco peroxidase, a mediator substrate (ABTS), and a second substrate. The approach is based on the establishment of a pseudo-steady-state concentration of the ABTS oxidation product in the course of co-oxidation with a poor substrate. A mathematical description of the experimental curve shape has been proposed to linearize the kinetic data and estimate the rate constant for such non-enzymatic interaction. The rate constants calculated from the steady-state kinetics for the non-enzymatic interaction of ABTS oxidation product with phenol and resorcinol were 360 +/- 40 and 770 +/- 60 M(-1).sec(-1), respectively. The values obtained have the same order of magnitude as the rate constant for ABTS oxidation product interaction with veratryl alcohol, calculated from electrochemical measurements (170 M(-1).sec(-1)) by Donal Leech's group. However, the kinetic curves for co-oxidation of ABTS and veratryl alcohol catalyzed by tobacco peroxidase exhibit a pronounced lag-period, which either points to the high rate of the non-enzymatic interaction between ABTS oxidation product and veratryl alcohol and thus, contradicts the electrochemical calculations, or indicates an enzymatic nature of the co-oxidation phenomenon in this particular case.