T H E
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PHYSICAL CHEMISTRY Registered in U.S. Patent Office 0 Copyright, 1975, by the American Chemical Society ~~~~
VOLUME 79, NUMBER 10 MAY 8,1975
Kinetics of Reduction of the Catalase-Hydrogen Peroxide Complex by Ethanol Mordechai .L. Kremer Department of Physical Chemistry, The Hebrew University,Jerusalem, lsrael (Received September 72, 1974) Publication costs assisted by the Central Research Fund of the Hebrew University
The effect of donors (alcohol) on the kinetics of the enzyme-substrate complex (ES) of catalase and H202 has been investigated theoretically. It has been shown that low concentrations of the donor affect only the terminal stage of the kinetics by causing the otherwise stable complex to disappear. By increasing the concentration of the donor, a t constant initial enzyme and substrate concentrations, the maximal concentration of the complex is also affected (decreased). The bimolecular rate constant of the reaction of ES with alcohol has been calculated by constructing simulated decay curves with the help of a computer and by comparing them with the experiment: k 5 = (1.00 f 0.05) X lo3 M-l sec-l. The “spontaneous’) decomposition of ES is discussed and it is shown that it may be due either to the reduction of ES by a residual donor in the system (exogenous or endogenous) or to an irreversible transformation of ES into ES’ (inactive). Some evidence favoring the latter possibility is discussed. The reactions of the enzyme substrate complex (ES) of catalase and H202(S) with various hydrogen donors (HaA), such as, alcohol, formate, etc., have been studied extensively by Chance,lr2 and also by other^.^,^ It was observed that, when catalase was mixed with H202 in the presence of a hydrogen donor, the concentration of the enzyme substrate complex rose rapidly to a maximum, then declined, at a rate depending on the total concentration of the hydrogen donor (ao), to zero. In a limited range of donor concentrations the decay of ES was pseudo-first order, the pseudofirst-order rate constant being proportional to ao. The rate constant of the reaction of the donor with ES (kb)was evaluated by using the equation T,ff is the time necessary for the concentration of ES to decrease from its maximal value (p,) to 0 . 5 ~ Deviations ~ . from eq 1 were observed, however, when, at a constant initial enzyme and peroxide concentration, a0 was increased or decreased beyond certain limits. No quantitative interpretation of the rate data has been given in this extended region of donor concentrations.2Jj Recently, a new kinetic analysis of the peroxidatic mechanism of catalase action has been performed. It has been found that, in a system containing catalase and H202 only, the enzyme substrate complex ES is stable.6 In the presence of a reducing donor, this complex becomes labile and its concentration decreases eventually, toward the end of
the reaction, to zero. It is the purpose of the present work (1) to investigate in a systematic fashion the effect of a reducing donor on the kinetics of the enzyme substrate intermediate and (2) to define conditions in which the decay of ES is of first order.
The Peroxidatic Mechanism Our discussion is based on the peroxidatic mechanism of catalase action. It is given by the following set of reaction~:~~’
E + S A E S e - p x ES + S L E + O2 + 2H20 P ES
P
x
+ HzA
E + A
+
2H20
a
E denotes a haematin group of catalase. Small type letters denote concentrations. e is the total concentration of catalase haematin? If a is zero, the concentration of ES increases, in a given reaction, toward an upper limit as time tends to This final concentration is a function of the initial concentrations of catalase and of peroxide and is itself bound by an upper limit given by k l e / ( k l k4).l0 The addition of a donor changes basically the kinetics of the intermediate. The concentration of the intermediate no
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Mordechai L. Kremer
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, , I: Effect of Donor on the Maximal TABLE Concentration of the Intermediate Complexa
0 00 1.89 0 1.89 5.00 0.0368 275 50.0 1.88 50.0 0.473 150 500 1.85 500 1.88 90 5000 1.69 5000 4.42 50 a k l = 3.01 X lo6 M - l sec-I; k4 = 5.59 x 106 M-1 sec-1; k b = 1.00x IO3 M - l sec-l; e = 5.44 rM; xo = 12 F M . 0
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Figure 1. Formation
and decomposition curves of the catalase-H202 intermediate in the presence of various concentrations of alcohol: kl = 3.01 X lo6 W 1sec-', k4 = 5.59 X IO6 W 1sec-'. k5 = 1.00 X lo3 W 1sec-', e = 5.44 yM, xo = 12 pM, +o(A) = 0 yM, ao(B) = 5 pM, a&) = 50 yM, ao(D)= 500 yM, ao(E)= 5000 pM. longer tends to a final upper limit, but passes instead through a maximum (Figure 1). The time course of p is, in general, complicated. It depends both on the initial concentrations of the reactants and on the relative magnitudes of the various rate constants. The kinetics of the declining phase of p , however, is greatly simplified in the real case of catalase, in which ks, for all known donors, is by at least three orders of magnitude smaller than either kl or k4. Because of the condition kg
