The reaction between aminotriazole and catalase

414

BIOCHIMICA ET BIOPHYSICAACTA

THE REACTION BETWEEN AMINOTRIAZOLE AND CATALASE P. NICHOLLS Molteno Institute, University of Cambridge (Great Britain) (Received September 29th, 1961)

SUMMARY I. Aminotriazole, which produces an irreversibly inhibited enzyme by reaction with catalase compound I, forms a typical ionic complex with free ferric catalase. It catalyses the reduction of compound II by endogenous hydrogen donor in a similar manner to other ionic ligands. 2. Unlike other ionic ligands, aminotriazole fails to accelerate the reduction of compound I to compound II. The steady-state level of compound I I thus remains low in the presence of aminotriazole in systems containing continuously generated peroxide. 3. Aminotriazole does not act as a hydrogen donor for any coupled oxidation in the presence of catalase. 4. Irreversibly inhibited catalase, formed in the reaction of aminotriazole and compound I, reacts slowly with cyanide, nitric oxide, and peroxides. Its reduced catalatic activity seems to be due to a much smaller rate of formation of compound I. 5. It is suggested that the irreversible inhibition may be due to a peculiar intramolecular reaction involving an otherwise inactive but reversible compound I - a m i n o triazole complex. 6. The spectrum and behaviour of irreversibly inhibited catalase are similar to those of other catalase preparations in which the protein moiety has been modified. A knowledge of the site of inhibition in the protein could provide important information about the relation between haematin and protein in this enzyme.

INTRODUCTION In 1955, HEIM, APPLEMAN AND PYFROM1 found that liver catalase activity was reduced in animals injected with AT. The activity of the enzyme in vitro was only inhibited at higher AT concentrations, and unlike the in vivo effect, was reversible. Subsequently, MARGOLIASHAND NOVOGRODSKY2 showed that the in vivo effect could be imitated in vitro by the addition of a system generating peroxide slowly. This irreversible inhibition is produced by a reaction between AT and catalase compound I (see ref. 3); a non-inhibitory reaction with catalase compound II is also promoted by the reagent s, 4 Abbreviations: AT, 3-amino-I,e,4-triazole; compound I, catalase peroxide compound I; compound II, catalase peroxide compound II; catalase-AT complex, the reversible compound of .AT and catalase; ATi-catalase the irreversible compound of AT and catalase. Biochim. Biophys. Acta, 59 (I962) 414-42o

AMINOTRIAZOLE AND CATALASE

415

The inhibited catalase formed in the reaction is spectroscopically distinct 2 and does not react readily with ligands of catalase such as cyanide 3. At least part of the AT seems to have combined with the protein moiety 3. In consequence of work on related compounds, MARGOLIASH et al. 3 concluded that the grouping involved in the reaction is > N . N H . C ( N H 2 ) : R (R = O, N, S); thus semicarbazide slowly produces the same effect. The analogy with the inhibition by azide 5 and the importance of any catalase reaction involving the protein, induced an investigation of the AT effect during a survey of catalase chemistry 6. The present paper describes the findings and suggests a partial mechanism. METHODS

Catalase was prepared from horse liver b y the method of KEILIN AND HARTREE7. The aminotriazole used was a gift from Dr. APPLEMAN to Professor KEILIN. Cysteine was once recrystallized as the hydrochloride. Notatin (glucose oxidase) was from the sample described by KEILIN AND HARTREE11. The reactions were followed spectrophotometrically in a Hilger Uvispek instrument with a glass prism. For the coupled oxidation experiments, Barcroft-Warburg microdifferential manometers were used at 3 °0 with air as the gas phase. RESULTS

Reaction of aminotriazole with free catalase

The reversible complex between AT and ferric catalase, postulated b y HEIM et al. ~ and b y MARGOLIASHet al. 3 to explain their kinetic data, has been observed b y direct spectroscopy in the visible region. I t has a spectrum of an ionic type similar to that of azide-catalase, with the 622-m/~ band shifted to 620 m/, and heightened (see Fig. 2 below). The dissociation constant at 20 ° and p H 5.7 appears to be 6. IO-z M, and the complex is unaffected b y a shift of p H from 5-4 to 6.8. Reaction of aminotriazole with compound I I

The secondary peroxide compound of catalase (compound II) was produced by reaction with peroxide continuously generated in the oxidation of glucose b y notatin s,°. In the presence of aminotriazole the rate of formation of compound I I was the same as in its absence, but the concentration of compound I I in the steady state was markedly reduced (Fig. I). This can be attributed to the action of the AT on the decomposition of compound II, also reported by MARGOLIASH AND NOVOGRODSKY4 for ethylhydroperoxide compound II, and illustrated in the reaction curve B of Fig. I. In promoting the decomposition of compound I I without affecting the steady state of compound I, aminotriazole is behaving in a remarkable manner. Hydrogen donors ° and anionse, 1° usually accelerate both the formation and decomposition of compound II. The exceptions, nitrite °, formate TMand azide 1°, react rapidly with compound I as multi-electron donors. According to MARGOLIASHet al. a, the reaction between AT and compound I I is second order, as is the reaction with phenols 9. But anionic ligands catalyse the decomposition of compound I I according to Michaelis-Menten kinetics TM. The rates of this reaction were therefore investigated using the compound I I produced in the presence of notatin and glucose. The results are given in Table I. Biochim. Biophys. Acta, 59 (1962) 114-42o

416

P. NICHOLLS

A 0.30

85 lamolcs

IOO°/o11-

.......

AT

O.261 8 O. 2~

I

Nototin Glucose

0

Fig. 4/zg liver AT;

I

I

5

I0

I

I

IS Minutes

20

1. The effect of AT on the s t e a d y state of c o m p o u n d I I in the n o t a t i n - g l u c o s e system. n o t a t i n and I m g glucose in solution added to catalase solution (final concentration 1.5. lO-5 M catalase h a e m a t i n in 2. 5 ml p h o s p h a t e , p H 5.4). Curve A, the solution contained 3-4" lO-2 M Curve B, control; AT added at I3. 5 min as indicated. T e m p e r a t u r e , 20 °. Measurements at 565 m/~, I-cm glass cell. TABLE I DECOMPOSITION OF COMPOUND I I BY" AMINOTRIAZOL• A T concentration (M)

Apparent velocity constant ( M - ' sec t)

0.03 o.o8 o.I6 0.32

0"25 °"15 o.14 0.07

Calculated parameters*

]

Km Ka

= o.i6 M = 0.06 M

kmax = 0.04 sec -1

* p H 5-7, 20°, 0.08 M phosphate. C o m p o u n d I I produced f r o m liver catalase by n o t a t i n and glucose.

The data suggest that although the reaction at low AT concentrations is second order, there is a limiting velocity at high concentrations similar to that found with other anions 1°. A complex with compound II would be expected to promote the decomposition in this manner, as shown in Fig. 3 below. Furthermore, the velocity constant for the apparent second-order reaction decreases from 0.26 M -1 sec -1 at pH 5-4 to 0.08 M -1 sec -1 at pH 6.9, a pH dependence characteristic of an endogenous donor reaction 1°, 13. In order to determine whether the lack of normal reactivity with compound I was due to a donor reaction, as in the case of azide 5, coupled oxidation n experiments were carried out. Not only does AT not act as a donor for the system, it inhibits the coupled oxidation of ethanol and ferrocyanide by producing the irreversibly inhibited form of catalase, ATi catalase (Table II). It was concluded that AT engages in no donor reactions with catalase, that like •all other ligands forming ionic complexes with the free enzyme it catalyses the decomposition of compound II, but that unlike most such ligands it does not promote the transition from compound I to compound II. B i o c h i m . B i o p h v s . Acta, 59 (I962) 414-42o

417

AMINOTRIAZOLE AND CATALASE TABLE II EI~FECT Or AMINOTRIAZOLE ON COUPLEI) OXIDATION

20 /~g notatin, 7.6 m M glucose, 30/~M catalase, o.25 M AT (when used), in p H 6 p h o s p h a t e buffer I ml total volume. Microdifferential m a n o m e t e r w i t h o u t alkali p a p e r s s h a k e n in air a t 3o°. A T absent

A T presog

Additions

None 2o m M E t O H 14 m M ferrocyanide

% utilisation*

Final spectrum

% utiUsa~ion*

o IOO

Compound II Free catalase

o 55

22

Compound II

o

Final spe~rum

ATi-catalase Free catalase + ATi-catalase ATi-catalase

* Percentage of generated H~O 2 used peroxidatically.

Formation and properties of the irreversible A T derivative (A Ti catalase) The results of MARGOLIASH et al. 3 were confirmed. An inactive derivative of catalase is produced in the presence of aminotriazole by all systems forming catalase compound I; notatin and glucose (Table II), ethyl hydroperoxide, and cysteine were among those employed. Fig. 2 compares the spectrum of the product of the reaction involving cysteine with that of the catalase-AT complex. i

I

i

£mM

t

t

I

'/' '

AT-CAT

IO

ATiCAT ,' // ,1//

CATALASE : AMINOTRIAZOLE

DEI~IVATIVES

0700 (=pI Fig. 2. The spectra of the reversible a n d irreversible c o m p o u n d s of AT a n d catalase. *ram calculated f r o m m e a s u r e m e n t s on 2.1- Io - s M catalase in I-cm glass cuvette~. A T - c a t a l a s e c o m p l e x (AT-CAT) formed w i t h o.6 M AT in p H 5.7 p h o s p h a t e ; irreversible A T - c a t a l a s e (ATiCAT) formed b y incubation with cysteine + trace of copper s u l p h a t e for 2 h.

The a-band of the irreversible derivative has shifted to 634 m~, while that of the reversible derivative is displaced to 62o m~. The 540 ml* band is of increased intensity in ATi catalase, but of decreased intensity in the catalase-AT complex. The reaction to form ATi catalase may therefore be followed by measuring the change in the 640-62o m~ difference spectrum. In this way a velocity constant for the cysteineB i o c h i m . B i o p h y s . A a a , 59 (I962) 414-420

418

p. NICHOLLS

induced transition (compound I ---+ ATi catalase) of 0.04 M - ' sec -1 at 0.025 M AT was obtained. This m a y be compared with the value of 0.025 M -1 sec -1 calculated by MARGOLIASHet al. ~ from the rate of decrease in catalatic activity. The inhibited enzyme has a small but definite catalatic activity; about 3 °Jo of the original rate was found in the present experiments, and the loss of activity curves given in ref. 3 suggests a similar value. The rates with which ATi-catalase reacts with complex-forming ligands are also very low. Only small spectroscopic changes are produced by fluoride and by azide; the cyanide complex takes 2-3 min to form from IO m M cyanide; the nitric oxide complex required a rather shorter period for full formation from nitrite in acid solution. No change could be observed with ethyl hydroperoxide; but the addition of sodium azide followed by hydrogen peroxide gave rise to the spectrum of nitric oxide ferrocatalase 5, indicating that all the enzyme remained active, but that the rate of formation of compound I was greatly diminished. The occurrence of these reactions differentiates this derivative from other pseudoirreversibly inhibited forms of the enzyme, such as NO. ferrocatalase 5 and hypophosphite-catalase 1°, in which the iron forms a complex with the inhibitor. The view of MARGOLIASH,that the inhibitor is bound to the protein moiety 3, is thus supported.

DISCUSSION MARGOLIASH et al. 3 employed a kinetic analysis 13 of their results which assumed a bimolecular reaction between compound I and aminotriazole, although they recognize the possible existence of catalase compound I - A T complexes. The occurrence of a maximum velocity for AT inhibition is attributed to the formation of the A T - c a t a l a s e complex at high AT concentrations; they calculate an equilibrium constant close to the value of 60 m M found in the present study by direct spectroscopy. An equation of the Michaelis form is also obtained, however, from a mechanism involving: (a) the formation of a reversible c a t a l a s e - A T complex (b) the formation and decomposition of compound I in the catalatic reaction (c) the formation of a reversible catalase compound I - A T complex, catalatically inactive but (d) producing ATi-catalase by a monomolecular reaction. Fig. 3 gives the reactions postulated, including the reaction with compound I I described above. The rate calculated for "compound I.AT --+ ATi catalase" given by kt is of the same order of magnitude as the uncatalysed "compound I --+ compound I I " reaction. It is much less than the "compound I --+ compound I I " reaction rate obtained in presence of a ligand such as fluoride 1°. If the given mechanism is correct, the reason for this inhibition of compound I I formation compared to ATi catalase formation is obscure. The p H independence of the inhibitory reaction 3 is in accord with the pH independence of the formation of A T - e n z y m e complexes (this paper). The similar ratio of the rates of compound I I decomposition and catalase inhibition in the cases of aminotriazole and S-methylthiosemicarbazide 3 suggests that the two reactions are related, as the common step of complex formation (Fig. 3) proposes. The strong temperature dependence of the inhibitory reaction, and its prevention by ferricyanide *, however, show that the system is more complicated than Fig. 3 suggests, ki at least Biochim. Biophys. Acta, 59 (x96"-')414-4zo

AMINOTRIAZOLE AND CATALASE

419

being a multiple constant for several reactions. These and other questions, such as the reason for an increase in rate of inhibition with time 3, remain unanswered. ATi catalase shows some spectral analogy to other derivatives of catalase in which the protein has been modified, Thus catalase under alkaline conditions (above Catalase-AT

/ AT ~aa--~

C a t a l a s e (Fe 3+) kl

A T i - c a t a l a s e <-

k~

Compound I-AT

/AT ~

+

Compound I

lk 7

/ AT Compound II-AT ~

, k4 t Catalase-AT

HzO ~

Compound II [

[ k4 / AT Ka / C a t a l a s e (Fe 3+)

Fig. 3. R e a c t i o n s b e t w e e n c a t a l a s e a n d a m i n o t r i a z o l e , k x = 6. Io e M - 1 sec -1, kq = lO -2 sec -x, h 4 = 6. IO- i sec -1, k4" = 4" lO-2 sec-1, Ka = o.06 M, Km = o.16 M, k, ~ 4" 1°-3 sec -1 (estim a t e d ) . T e m p e r a t u r e 2o °, p H 5.4.

pH Io) and freeze-dried "reducible" catalase 14 both show the shift in a-band towards the infrared. In these cases also, catalatic activity is diminished b y a decrease in the value of kl (the combination of enzyme and peroxide). But the changes involved are different. Thus reducible catalase can be further inhibited b y incubation with AT and H,O2. And ATi catalase is only slightly more reducible than native catalase; standing for several hours in presence of dithionite under carbon monoxide was required for the production of CO-ferrocatalase. ATi catalase and the analogous derivatives provide a unique opportunity for the investigation of an active protein site in catalase. On present evidence, it is not possible to determine the amino acid residue reacting with > N . N H . C ( N H 2 ) : R (plus perhaps the two oxidizing equivalents of compound I). The mechanism of Fig. 3 emphasizes the difficulty of discovering this indirectly, because it implies that two quite different properties can affect the velocity of the inhibitory reaction: (a) the equilibrium constant for the reaction of the AT analogue with compound I (Kin of Fig. 3) ; (b) the rate of the monomolecular transition (kt of Fig. 3).

ACKNOWLEDGEMENTS

This work was carried out while the author was in receipt of a scholarship for training in research methods from the Medical Research Council of Great Britain. He would also like to express his appreciation of Professor KEILIN'S advice and encouragement, and his thanks to Dr. MARGOLIASH for several discussions as to the mechanism of the reaction described.

Biochim. Biophys. Acta, 59 (1962) 414-42o

420

P. NICHOLLS REFERENCES

1 W. G. HEXM, D. APPLEMAN AND H. T. PYFROM,Science, 122 (1955) 693. 2 E. MARGOLIASH AND A. NOVOGRODSKY,Biochem. J., 68 (1958) 468. s E. MARGOLIASH, A. NOVOGRODSK¥ AND A. SCHEJTER, Biochem. J., 74 (196o) 339. 4 E. MARGOLIASH AND A. NOVOGRODSKY, Bioehim. Biophys. Acta, 3 ° (1958) 182. s D. KEILIN AND E. F. HARTREE, Nature, 173 (1954) 720. 6 p. NICHOLLS, Ph. D. Thesis, Cambridge University, 1959. D. KEILIN AND E. F. HARTREE, Biochem. J., 39 (1945) 148. s B. CHANCE, Biochem. J., 46 (195o) 387. 9 D. KEILIN AND P. NICHOLLS, Biochim. Biophys. Aeta, 29 (1958) 302. 10 p. NlCHOLLS, Biochem. J . , 81 (1961) 365. 11 D. KEILIN AND E. F. HARTREE, Biochem. J., 60 (1955) 310. 12 B. CHANCE, J. Biol. Chem., 194 (1952) 471. 13 E. MARGOLIASH AND A. SCHEJTER, Biochem. J., 74 (196o) 349. 14 A. L. DOUNCE AND R. R. SCHWALENBERG, Science, i i i (195o) 654.

Biochim. Biophys. Acta, 59 (I962) 414-42o

MITOCHONDRIAL

AND CYTOPLASMIC THIAMINE

BAKER'S DECLINE HEIKKI

IN

YEAST DURING THE

OF DECARBOXYLASE

ACTIVITY

S U O M A L A I N E N AND S A K A R I R I H T N I E M I

Research Laboratories of the State Alcohol Monopoly (A lko), Helsinki (Finland) (Received October 2nd, 1961 )

SUMMARY

Mitochondrial and cytoplasmic fractions of baker's yeast were prepared by mechanical disintegration from cultures grown rather anaerobically and in vigorously aerated conditions. The mitochondrial fraction contained only 25-26 % of the succinic acid dehydrogenase activity and the succinic acid oxidase activity. The total thiamine content of the mitochondria (including the disrupted ones) amounted to 6.0 t*g for the anaerobic cultures and for the aerobic cultures to 5.I ~g per g fresh yeast. The total content of thiamine in the cytoplasm was found to be 1.5 t~g at the anaerobic stage and a scant 0.3 t~g per g fresh yeast at the aerobic stage. 80-95 % of the thiamine is located in the mitochondria. During transfer from anaerobic to aerobic culture conditions, the mitochondrial content of thiamine decreases b y a scant 20 %, whereas the cytoplasmic content falls by a good 80 %. This sharp decrease in the thiamine content of the cytoplasm is consistent with the earlier observation that the decarboxylase activity of baker's yeast strongly decreases on transference from anaerobic to aerobic culture conditions.

Biocl~im. Biophys. Acta, 59 (1962) 420-425