Peroxidase (EC 1.11.1.7) catalyzes the oxidation of
electron donors of various chemical structure with hydro-
gen peroxide. If not accounting for the formation of
Michaelis-type complexes [1], the formal description of
the peroxidase catalytic mechanism is represented by the
system of chemical reactions as shown in Scheme 1:
k
1
E + H
2
O
2
→ EI + H
2
O,
k
2
EI + S → EII + P,
k
3
EII + S → E + H
2
O + P,
Scheme 1
where E, EI, and EII are the native enzyme and its
Compounds I and II, respectively, and S and P are a
substrate and the product of its one-electron oxida-
tion.
The Michaelis complexes are usually omitted
because they cannot be detected for native peroxidases by
pre-steady-state methods, although these complexes have
been observed for some mutant forms [2, 3].
The phenomenon of substrate–substrate activation
is well known in peroxidase catalysis [4, 5]. This type of
activation manifests itself by the stimulated oxidation of a
poorly or non-oxidized (“bad”) substrate (S
1
) in the pres-
ence of an easily oxidized (“good”) substrate (S
2
). Three
different mechanisms can describe substrate–substrate
activation. In the first case, Compound I is active towards
a “bad” substrate, while Compound II does not oxidize it
(Scheme 2):
Biochemistry (Moscow), Vol. 68, No. 9, 2003, pp. 1006-1011. Translated from Biokhimiya, Vol. 68, No. 9, 2003, pp. 1231-1237.
Original Russian Text Copyright © 2003 by Hushpulian, Fechina, Kazakov, Sakharov, Gazaryan.
0006-2979/03/6809-1006$25.00 ©2003 MAIK “Nauka / Interperiodica”
* To whom correspondence should be addressed.
Non-Enzymatic Interaction of Reaction Products
and Substrates during Peroxidase Catalysis
D. M. Hushpulian
1
, V. A. Fechina
2
, S. V. Kazakov
3
, I. Yu. Sakharov
1,4
, and I. G. Gazaryan
1
*
1
Department of Chemical Enzymology, School of Chemistry, Lomonosov Moscow State University, Moscow 119992, Russia;
fax: (7-095) 939-5417; E-mail: igazaryan@hotmail.com
2
Bach Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow 117032, Russia;
fax: (7-095) 954-2804; E-mail: zherdev@inbi.ras.ru
3
Department of Chemistry, Chemical Engineering and Materials Science, Polytechnic University,
6 Metrotech Center, Brooklyn, NY 11201, USA; E-mail: skazakov@msn.com
4
Department of Chemistry, Plekhanov Russian Economic Academy, Stremyannyi Pereulok 28, Moscow 113054, Russia;
fax: (7-095) 237-9342; E-mail: sakharov@enz.chem.msu.ru
Received October 24, 2002
Abstract—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 establish-
ment of a pseudo-steady-state concentration of the ABTS oxidation product in the course of co-oxidation with a poor sub-
strate. A mathematical description of the experimental curve shape has been proposed to linearize the kinetic data and esti-
mate 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 M
–1
·sec
–1
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 prod-
uct and veratryl alcohol and thus, contradicts the electrochemical calculations, or indicates an enzymatic nature of the co-
oxidation phenomenon in this particular case.
Key words: tobacco peroxidase, mediator, second-order rate constant, phenol, resorcinol, veratryl alcohol