ISRAEL JOURNAL OF CHEMISTRY

Vol. 8, 1970, pp. 799-807

INHIBITION OF CATALASE BY CYANIDE

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

The cyanide inhibited decomposition of HP2 by catalase obeys the rate

equation

(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.,

The Kinetics of Inhibition

Competitive (a), uncompetitive (b), mixed (c) and non-competitive (d)

types of inhibition have been defined in relation to the simplified mechanism
for enzyme catalysis
1 3
E + S~ ES -E + P (A)
2

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)

cf> = Ki (1 + ~]) uncompetitive inhibition (3)

cf> = K [S] + KM mixed inhibition (4)

1 K'
[S]jtt-+ K M
1

4> =K i noncompetitive inhibition (5)

C/J = [I)-!L, K - k2 + ks
v - vi M - k
1

It follows from these equations that (1) in

6 itself, does not constitute proof that the
inhibition is incomplete. Under suitable
circumstances, C/J can become independent
5 of [8] for any type of inhibition. In order
to decide between the different possible
4 cases a knowledge of the relative magni-
tudes of [8] and of Km is required.
4>3 For the catalatic reaction a value of
K M " 1,1M [pH = 4,3-10,0 andt = 30OC)
., has been established [7,8), superseding
92 an earlier incorrect value of 25 mM [6).
•
~
The independence of C/J of [H:P2] at [H 20 2)
< 0.1 M thus excludes only uncompetitive
I 0
3».
inhibition (Eq. By increasing [Ha02]
[7]. Therefore the inhibition is competi-
tive. The kinetics ofthe inhibition follows
2 ~ 4 5 Eq. (2) and is therefore definitely com-
1i
L~02J M petitive. *
FIG. 1... asa function of (HzOtl. 0.12 /1M horse liver This result does not imply, however,
catalase, pH = 7.0, t = 2S"C. Data of Ogura [7]. that the only reaction of cyanide in the
" Theresults also agree with Eq, (4), provided that K'i »K i. Under these circumstances, however, mixed
and competitive types of inhibition become indistinguishable.
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Vol. 8, 1970 INHIBITION OF CAT ALASE

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

E represents catalase haematin.

There are several possibilities of postulating inhibitory reactions of
CN - in agreement with Eq. (2).
These include:
a) Reaction of CN- with E in either mechanism B,C or D.
Kj
(E + CN- ~ECNT
b) Reaction of CN- with CI in mechanism B or C, or reaction with C n
K.·
in mechanism D. (CI (Cll) + CN- ~ CI·CN- (Cu' CN-»
c) Reaction with both E and C 1 in mechanism Band C or with both
E and c» in mechanism D (Ki 1= Kp.
d) The same as (c) but with Ki = Kj.
Reactions involving C; in mechanism Band C or C 1 in mechanism D lead
to different kinetics.

801
 M. L. KREMER Israel J. Chern.,

The resulting rate equations are:

Case a: if! = (l + ~)Ki (l + [H~al) ( 6)

Case b : ¢ = (l + ~Kl (1 + [H~Oal) (7)

{3+Q 1)
Case c: cf. = K. Ki (1 + [HPa (8)
I y
J3 + Q-

K!1

d ct· = K·I (1 + [HPa])

C ase: (9)
y

The expressions for the constants Q, J3 and y in terms of the rate

constants relating to the various mechanisms, are summarized in Table I.

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

1/1 0 is the limiting value of ¢ for [HP2] «Y.

In all the reported cases epo > K]. In terms of Eq. (8') this implies that
K i < K i ' . ¢o calculated on the basis of Eq. (8') lies thus between K i and
(1 + ;)K i . Considering the two extreme cases (6') and (9') it is evident that
a large difference between ¢o and K; would favour (6'). On the other hand,
if a< (3, the distinction between Eqs. (6') and (9') and to an even greater
extent, the distinction of Eq. (8') from (6') and (9') becomes difficult.
Chance reported cPo = 4.7 ~M and Ki = 4uM for horse blood catalase at
pH = 7 and t = 25°C [1). A larger difference of <to= 7.97 ± 2~}\'i and K, =
4.60 ± 1.36 ~M at 13.6 ± 2°C have been reported by Wolfe et a1. for
bacterial catalase [13]. These differences have been interpreted in terms
of Eqs. (6') and (8') respectively [12,13]. Since, however, the observed
differences are rather small and since the values of a] {3 used are not all
well established, * additional evidence for the mechanism of inhibition is
desirable. Also Eqs. (6'), (8') and (9') are not strictly applicable to the
results of Wolfe et al ., since the fraction of bound cyanide in their kinetic
experiments was not negligible [14].

Measurement of the Concentration of Cyanide Complexes

A different approach to the study of the mechanism of inhibition is provided

by the direct measurement of the concentration of the various species which
are formed in the inhibited reaction mixture. Chance [15] has measured
the differential optical density of a mixture of catalase, cyanide and HP2
against a pure catalase solution of equal strength at 435 nm where catalase
and the catalase-HP2 complex are isobestic. He denoted it by ~2' The
differential optical density of an identical solution of catalase and cyanide
but without HP2 as against pure catalase was denoted by ~1' Assuming the
formation of a single complex E .CN - in the system, the degree of saturation
of catalase by HP2 could be calculated [9]. Qualitative and quantitative
discrepancies from the predicted behaviour remained, however.
Using a more general treatment, the formation of two cyanide complexes
can be assumed in the system. In this way it is possible to treat all the
significant cases discussed in the kinetic section.
The equations will be developed on the basis of the original pe'roxidattc
theory of catalase action
1
- CI
(E)
C I + H 202 .i E + O 2 + 2 Hp
which is the low [HP2] extension of mechanism C. With the help of Table I,
however, the results can be converted to the equivalent equations of
mechanism B or D.
• Ot/B = kl/~ = 0.67 has been used by Wolfe et al, for bacterial catalase [17]. From the results of pte-
steady state measurement, however, 5.4 or 0.18 can be calculated for the same quantity [9] (Data of Table II
in [2]). (The two values correspond to the roots of a quadratic equation.)

803
 M. L. KRF.MER Israel J. Chern.,

Assuming the formation of the species E . CN - and C I' CN - in the

inhibited system, the ratio of the differential optical densities at 435 nm
becomes

~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:

ICI Jz + ICI . CN-la I_f~[ECN-h

[E]" ~ [E)o'
t

(11)

The subscript 0 denotes total concentration.

The dependence of the reciprocal degree of saturation by H 20 2 on the
concentration of free cyanide is given by

The concentration of free cyanide in Eq. (12) is calculated from

[CN-)2 = [CN-]o- (1 +~ Ki,).A2... f (E· CN-It (13)

k. K j ~l

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

0.4 0.8 2.0 4.0 8.0 20 50

0.56 0.62 0.73 0.70 0.80 0.82 0.92
[E 'CW]t 11M 0.18 0.35 0.79 1.35 2.04 2. 76 3.13
[EJe/([CI]Z +[C1CN-]z) 1.67 1.90 2.62 2.59 3.96 4.55 8.26
Ki + [CN-Jz
0.107 0.113 0.131 0.164 0.224 0.376 0.587
K( + [CN"]z

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

In one series of calculations K i I (aasumed) was varied while g was kept

constant. The results of these calculations for g = 1 are shown in Fig. 2
and Table III.

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.

Ki' (assumed) IJM 40 20 13,3 10 8

K( (obtained) IJM 23 14 11 9.8 9.5

g (assumed) " 1

805
 M. L.KREMER Israel J. Chern.,

This procesure yielded a self consistent value of K]". The intercept

of the self consistent plot, however, fell below the theoretical value of
unity (Fig. 2). Similar results were obtained when the value of g was
varied. Table IV summarizes the results of these calculations.

TABLE IV.

s (assumed) 2 1 0.5 0.2 0.1

Ki ' (self consistent) 11M 6.0 ]0 ]8 25 30

Relative deviation of
intercept -1.84 -0.54 -0.23 -0.17 -0.14

l:1~1 0.39 1.3 2.8 33 34

By decreasing g, K i' (self consistent) showed a gradual increase. The

relative deviation of the intercept (defined as the difference from unity
divided by the span of the values of [E]o/([Cd a + [CI . CN-l a)) decreased with
decreasing g. The deviation of the points from the "best straight line",
however, showed a rapid increase with decreasing g. (6= deviation of the
reciprocal degree of saturation from the best straight line in the graphical
plot of Eq. (12)). The region where both errors were simultaneously small
extended between g = 0.5 and g = 1. From here Ki' = 14 ± 4IAM is estimated.
This value agrees well with Kif = 10/AM calculated by Beers [121 from
kinetic data using <Po = 4.7/AM and K i = 4/AM (horse blood catalase, experi-
mental data of Chance [1]).
The reason for negative deviation of the intercept may be due to several
factors.
1) A different value should have been used for k t/k 4 •
2) Insufficient [HaG a] in the experiments to ensure the establishment
of a true steady state.
A variation of k 1/k4 within reasonable limits had no appreciable effect
on the results. Taking k t/k 4 = 0.715 and g = 1, the relative deviation of the
self consistent plot became - 0.36 while Kit increased to l2/AM.
The estimation of the second factor is more difficult. Insufficient [HPa].
increases the reciprocal degree of saturation.

An increase of [El 2/[Cd2 causes an increase of [E]o/([Crb + [CICN-h).

The effect is more pronounced at high [CN-] since K i ' > Kj , It results in
an increased slope and, possibly, in a decreased intercept. Chance
observed a decrease of 6 2/ til from 0.70 to 0.66 by increasing [HPal from
10/AM to 100-1000 lAM ([CN-]o = 4/AM, tables 1 and 2 in [15]). It indicates
a partial conversion of E·CN- to the less strongly absorbing species CICN-
It also shows a decrease of the reciprocal degree of saturation by HaG a
when steady state conditions are approached. No data exist for other
concentrations of cyanide. Therefore the correction of all the results for
this effect is not possible.

806
Vol. 8, 1970 INHIBITION OF CATALASE

It is concluded from the preceeding discussion that cyanide combines

in a reversible manner with both the free enzyme E and the enzyme-
substrate complex CI. Both reactions contribute to the inhibiting effect
of cyanide; the enzyme substrate complex has, however, a lower affinity
towards CN - than the free enzyme. The formal type of Inhibjtion is
competitive. The actual mechanism is, however, more complicated than
the classkal competitive type. It is due to the dual role of H 20 2 in both
forming and decomposing the intermediate complex.
Equations (11) and (12) are not suitable for the determination of the degree
of saturation of catalase haematins by peroxide when no cyanide is present.
In fact, the knowledge of the parameter k 1/k4 = [C 11 [Elis needed for the evalua-
tion of the quantity [Elol ([C r 12 + [CrCN-1 2) and for the determination of K, I . An
exception is provided by the case when K i = K i I . Equation (12) then becomes

[E]o
( 12a)

The reciprocal degree of saturation is then independent of [CN -] and is

equal to its value in the absence of CN - The catalase r-eac tipn however,
does not conform with this case.
There is no indication that the binding of one haematin group by cyanide
influences the reactivity of the others towards H 20 2 • This is in accordance
with the fact that the haematin groups of catalase act independently [16).

ACKNOWLEDGEMENT

I am indebted to Professor Britton Chance for helpul discussions.

REFERENCES

1. B.CHANCE, J. Bioi. Chern •• 179, 1299(1949)

2. B.CHANCE, D.S.GREENSTEINandF.J.W.ROUGHTON, Arch. Biochim. Biophys., 37, 301(1952)
3. Ph. GEORGE, Advances in Catalysis, Vol. IV, 397(1952)
4. P.NICHOLLS, G.R.SCHONBAUM, in: P.D.Boyer, H.Lardy and K.Myrback, The Enzymes,
Academic Press, Vol. VIII, p. 201 (1963)
5. K.J.LAIDLER, T he Chern ical Kinetics of E nzym e Action, Oxford Univ. Press, p. 326 (1958)
6. R. K. BONNICHSEN, B.CHANCE and H. THEORELL, Acta Ch em. Sc and., 1, 68 (1947)
7. Y.OGURA, Arch. Biochem. Biophys., 67, 288 (1955)
8. P.JONES and A.SUGGETT, Biochem. J., 110,617 (1968)
9. M.L.KREMER, Biochim, Biophys. Acta, 198, 199(1970)
10. P.JONES and W.F.K.WYNNE-JONES, Trans. Faraday Soc., 58,1148(1962)
11. A.S.BRILL, in M. FLORKIN and E. STOTZ, Com pre he n s i v e Bio ch em i 'try, Vol. 14, Elsevier,
Amsterdam, p, 475 (1966)
12. R.F.BEERS, J. Phys. c h e ms, 59, 25 (1955)
13. A. D. WOLFE, R. F. BEERS, I. W. SIZER. Arc h. Biochern. Biop hy s ., 72, 353 (1957)
14. M.L.KREMER, Israel J. Chem., 5,137(1967)
15. B.CHANCE, J. BioI. Chern., 179, 1311 (1949)
16. MoL.KREMER, J. Theor. Biology (in press)
17. B.CHANCE and D.HERBERT. Biochern. J•• 46, 402(1950)

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