Non-Enzymatic Interaction of Reaction Products and Substrates in Peroxidase Catalysis

D. M. Hushpulian¹, V. A. Fechina², S. V. Kazakov³, I. Yu. Sakharov^1,4, and I. G. Gazaryan^1*

¹Department of Chemical Enzymology, Faculty of Chemistry, Lomonosov Moscow State University, Moscow 119992, Russia; fax: (7-095) 939-5417; E-mail: igazaryan@hotmail.com

²Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 117032, Russia; fax: (7-095) 954-2804; E-mail: zherdev@inbi.ras.ru

³Department of Chemistry, Chemical Engineering and Materials Science, Polytechnic University, 6 Metrotech Center, Brooklyn, NY 11201, USA; E-mail: skazakov@msn.com

⁴Department of Chemistry, Plekhanov Russian Economic Academy, Stremyannyi Pereulok 28, Moscow 113054, Russia; fax: (7-095) 237-9342; E-mail: sakharov@enz.chem.msu.ru

^* To whom correspondence should be addressed.

Received October 24, 2002

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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.

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KEY WORDS: tobacco peroxidase, mediator, second-order rate constant, phenol, resorcinol, veratryl alcohol

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