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M.L.Kremer
Johnsun Researcb Foundation,
University of Pennsylvania, Philadelphia
and
Department of Physical Chemistry,
The Hebrew University of Jerusalem
ABSTRACT
The kinetics of the inhibition of the catalatic reaction by cyanide is investigated. The
inhibition is formally competitive. Cyanide can combine with both the free enzyme and the
active enzyme substrate complex, the latter combination being more labile. The dissociation
constant of the enzyme-substrate- inhibitor complex for horse liver catalase has been calculated
from spectrophotometric data: Ki = 14 ± 4 pM. The agreement with the value calculated
from kinetic data by 8eers for horse blood catalase is satisfactory.
Introduction
(1) .
provided that [HP2 1 < 0.1 M [11. Vi and v are the rates of reaction in the
presence and absence of cyanide, other parameters being equal. Equation
(1) has been interpreted as indicating reversible, non -competitive
inhibition [2,3,4]. On the other hand, direct measurements of the extent
of formation of the cyanide complex in a reacting mixture of catalase and
H 20 2 have been interpreted in a way which implies that cyanide is not a
reversible inhibitor in the system [10, 15, 17}.
In fact it can be shown that
1) both sets of data are consistent with a single mechanism of inhibition,
2) the inhibition is reversible,
3) the inhibition is formally competitive.
4) both catalase and catalase-H 20 2 form inactive complexes with cyanide.
Received June 16, 1970
799
M.L.KREMER Israel J. Chern.,
They denote cases in which an inhibitor I is able to form the inactive com-
K'l K'l
plexes EI (E + I:: EI) (case a) or ESI (ES + I : ESI) (case b) or both of them
having either KiF K i (case c) or K i = KI (case d), E denotes the free enzyme,
S the substrate and P the product of reaction. The appropriate rate
equations (provided that both the substrate and the inhibitor are present in
a large excess over the enzyme) can be expressed in the form
cf> = K·1 (1 + KM
[SI) competitive inhibition (2)
C/J = [I)-!L, K - k2 + ks
v - vi M - k
1
This result does not imply, However, that the only r-eactrcn of cyanide
system is a competition with H 20 2 for free catalase haernatin . The reason
for it is that mechanism A does not apply to the catalatic reaction.
The catalatic mechanism has been shown to involve a step in which an
active catalase - H 20 2 complex reacts with a further molecule of H 20 2
(or with another acceptor if present) [2, 9}. The effect of substituting such
a reaction for step 3 of mechanism A is to eliminate altogether the Michaelis
constant KM from the rate equation [2}. In order to re-introduce it, one
of several alternative assumptions is necessary. One is to aasumethe
formation of a biperoxy complex of catalase haematin. This complex may
be catalytically active (mechanism B) or catalytically inactive (mechanism
C) [7}. An alternative possibility is to assume that two active monoperoxy
complexes are involved (mechanism D) [10,11).
1
E+HP2- CI
6
C 1 + H 202 ;:::
7
cr (B)
Ci 1. E + 02 + 2H 20
1
E +HP2-CI
C 1 + HP2! Ci (C)
7
4
C 1 + H 20 2 - E + 02 + 2Hp
1
E + HP2:: C 1
2
3
C 1 - ClI (D)
4
Cn + HP2 -E + 02 + 2Hp
801
M. L. KREMER Israel J. Chern.,
{3+Q 1)
Case c: cf. = K. Ki (1 + [HPa (8)
I y
J3 + Q-
K!1
TABLE I
~Iel"hanism ex B y
B kl
ka
~+K
K k1
~ k7
C" k1 ~ (1 +-,,-)--
k1 ks
klka kz~ka + ~
D ~
kz+k3 k1 ~
K (in B) = ka+k7
ks
• Applying the condition [HzOzJ« Y to mechanism C. Eqs, (6) through (8) yield the rate expressions for
inhihition previously obtained by Beers [12J.
'Yis the formal Michaelis constant of the reaction: for [HaO a] = y, v = v m a x/2.
Equation (7) can be excluded from further consideration since cyanide
combines with free catalase haematin. The remaining equations, (6). (8)
and (9) become for [HPa] « y
(6')
(8' )
(9' )
802
Vol. 8, 1970 INHIBITION OF CATALASE
803
M. L. KRF.MER Israel J. Chern.,
~t [E· CN-It
r;-= f [E' CN-h
(10)
g
f"CICN - - EE
~CN- -EE
The subscripts 1 and 2 refer to the two experiments of Chance [15]. Kj
and K i ' are the dissociation constants of E'CN- and CrCN- respectively
f" denotes extinction coefficients.
The degree of saturation of catalase haematins by H 20 2 in the presence
of cyanide is calculated from the expression:
(11)
For K i ' » [CN-la (when the formation of Cj : CN- can be neglected) Eq. (12)
becomes identical with Eq. (3) of [9].
Table II and Fig. 2 show the application of Eqs. (11) and (12) to the
experimental data of Chance [15]. The parameters K i = 4JLM (horse blood
= =
catalase, t = 25°C, pH 7.0 [lJ and k1ik4 0.455 (beef liver catalase, t =
t = 24 ± IOC, pH = 7.0 (9) were used in the calculations.
In the course of calculation a pair of values for g and K i ' was assumed
and the degree of saturation of haematins by H 20 2 was calculated at different
[CN -]0. The reciprocal degree of saturation was then plotted according to
Eq. (12). From the slope of the resulting straight line K i ' was calculated
and compared with the value assumed initially for it.
804
Vol. 8, 1970 INHIBITION OF CATALASE
TABLE II
Data of Chance fl5]. Horse liver catalase. [Elo " 3.4IJM, [HzOzJo" 10 IJM, pH " 6.5
kd~ " 0.455 Ki " 4.0 1J}.1 g (assumed) " I, Ki' (assumed) "40 1J}.1
6
.....e'
I
Z
5
0
00
.... ~ 4
UJ
~ +
N
-:. 3
0
~
03 04 05 06 0·7 08 09 1·0
Kj+[CN-]2
Ki +[CN-]2
FIG.2. The reciprocal degree of saturation by HzOz as a function of (Ki + [CN-]z)/(K i' +
[CN-]z). 3.41JM horse liver catalase haematin, [HzOzJ " 10IJM pH "6.5. Data of
Chance. [15]. g (assumed) "1. K'i (assumed) 1IM:8 (bl, 13.3 (-l. 40 (0),
TABLE In.
g (assumed) " 1
805
M. L.KREMER Israel J. Chern.,
TABLE IV.
Relative deviation of
intercept -1.84 -0.54 -0.23 -0.17 -0.14
806
Vol. 8, 1970 INHIBITION OF CATALASE
[E]o
( 12a)
ACKNOWLEDGEMENT
REFERENCES
807