ON THE MECHANISM OF ENZYME ACTION A STUDY OF THE DECOMPOSITION OF MONOETHYL HYDROGEN PEROXIDE BY CATALASE AND OF AN INTERMEDIATE ENZYME-SUBSTRATE COMPOUND BY KURT G. STERN (From the Department of Physiological Chemistry, Yale University, New Haven) (Received for publication, March 23, 1936) The classical methods of enzyme chemistry were of necessity restricted to the study of the catalysis as a whole. Either the rate of disappearance of the substrate or the rate of formation of the end-products was measured. The kinetics of these processes could in some instances be explained by the assumption that the catalyst combines with the substrate to form an unstable inter- mediate which may either reversibly dissociate into the two com- ponents or break down with the formation of free enzyme and the split-products (1, 2). The spectroscopic observation of the formation and the break- down of an intermediary enzyme-substrate compound during the action of catalase on monoethyl hydrogen peroxide (3) permits an experimental analysis of the two main phases of the enzyme reaction: Enzyme + substrate ti enzyme-substrate compound (1) Enzyme-substrate compound -+ enzyme + product molecules (2) Reaction 1 lends itself to optical study. The over-all process (Reactions 1 + 2) is measured by volumetric determination of the substituted peroxide. EXPERIMENTAL Preparation of Enzyme Solutions-Purified catalase solutions were prepared from horse liver* essentially according to Zeile and r The author is greatly indebted to Chappel Brothers, Inc., Rockford, Illinois, for generously supplying the frozen horse liver used for these preparations. 473 by guest on May 12, 2018 http://www.jbc.org/ Downloaded from
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ON THE MECHANISM OF ENZYME ACTION
A STUDY OF THE DECOMPOSITION OF MONOETHYL HYDROGEN PEROXIDE BY CATALASE AND OF AN INTERMEDIATE
ENZYME-SUBSTRATE COMPOUND
BY KURT G. STERN
(From the Department of Physiological Chemistry, Yale University, New Haven)
(Received for publication, March 23, 1936)
The classical methods of enzyme chemistry were of necessity restricted to the study of the catalysis as a whole. Either the rate of disappearance of the substrate or the rate of formation of the end-products was measured. The kinetics of these processes could in some instances be explained by the assumption that the catalyst combines with the substrate to form an unstable inter- mediate which may either reversibly dissociate into the two com- ponents or break down with the formation of free enzyme and the split-products (1, 2).
The spectroscopic observation of the formation and the break- down of an intermediary enzyme-substrate compound during the action of catalase on monoethyl hydrogen peroxide (3) permits an experimental analysis of the two main phases of the enzyme reaction:
Enzyme + substrate ti enzyme-substrate compound (1)
Reaction 1 lends itself to optical study. The over-all process (Reactions 1 + 2) is measured by volumetric determination of the substituted peroxide.
EXPERIMENTAL
Preparation of Enzyme Solutions-Purified catalase solutions were prepared from horse liver* essentially according to Zeile and
r The author is greatly indebted to Chappel Brothers, Inc., Rockford, Illinois, for generously supplying the frozen horse liver used for these preparations.
Hellstrijm (4). It was found expedient to carry out all operations of separation in a centrifuge which can accommodate four bottles of 500 cc. capacity each. Alumina gel (aluminum hydroxide, pure, moist (Eimer and Amend)) was used as adsorbent. Some data concerning the four enzyme solutions used in the present experiments are given in Table I.
For most of the experiments the undiluted enzyme preparations were used. These were dark brown in color and showed the typi- cal catalase absorption spectrum in layers ranging from 2 to 5 cm., depending on their enzymatic activity. The secondary sodium phosphate used for the elution of the enzyme from the alumina gel adsorbate was neutralized by adding solid primary
TABLE I
Data for Catalase Preparations
Cat&se No.
XXIX XXXIV xxxv XXXVI
Liver used Eluate obtained
gm. cc.
4,500 500 3,700 200 3,700 250
10,000 400
Activit.y*
/c 7125 2020 5300 4525 * I
* The activity was determined by measuring the rate of hydrogen peroxide decomposition by the highly diluted enzyme solution at 0”, p1-I 6.6, and at a total substrate concentration of 0.01 M. k is obtained as the product of the monomolecular velocity constant, calculated from the amount of substrate destroyed within the first 5 minutes of the experiment, and of the dilution factor of the enzyme.
potassium phosphate. The enzyme solutions were stored over chloroform in the refrigerator (approximately at +a”) and fil- tered before use.
Preparation of Substrate Xolutions-Solutions of monoethyl hy- drogen peroxide were prepared by following essentially the direc- tions given by Baeyer and Villiger (5) and by Rieche (6). The alkylation of hydrogen peroxide was found to give better yields if carried out with efficient cooling, the reaction temperature being kept near 10” by external cooling with an ice and water mixture. Throughout the alkylation process (requiring about 6 hours) and the subsequent neutralization with sulfuric acid, the reaction mixture was vigorously stirred with an electric stirrer.
The distillation of the substituted peroxide together with water was carried out under atmospheric pressure. The temperature inside the distillation flask was near loo’, whereas the oil bath was heated up to 150”. The distillation was discontinued after approximately one-half of the reaction mixture had been distilled over.
Since a sample of pure monoethyl hydrogen peroxide prepared according to the ether extraction method of Rieche and Hitz (7) gave essentially the same results as the dilute solutions obtained by single distillation, the distillates obtained as described above were used directly for the experiments after neutralization with solid secondary sodium phosphate. The amount of peroxide was determined by the iodometric method outlined below and was found to be between 1 and 2 M. These solutions were found to keep well for several months if stored in the refrigerator.
Volumetric Study of Decomposition of Monoethyl Hydrogen Peroxide by Catalase
Method
For the estimation of monoethyl hydrogen peroxide Baeyer made use of an iodometric method. According to Rieche (8), however, the active oxygen of this peroxide may not be quantita- tively determined with either hydriodic acid or titanous trichlo- ride. The latter author therefore carried out his analysis by oxi- dation with chromic acid; the acetic acid formed was distilled off in the presence of an excess of phosphoric acid and determined by acidimetric titration. This procedure is not only somewhat tedious but is also not applicable to solutions containing foreign organic matter. Inasmuch as the object of most of the experi- ments reported in this paper was to follow the breakdown of the substituted peroxide as catalyzed by the enzyme and to study the effect of temperature, pH, etc., on the rate of this reaction, it was not necessary to assay accurately the absolute concentration of the peroxide but only to record the relative changes in concentration with time. It was found that the iodometric method as used by the author for studying the breakdown of hydrogen peroxide by catalase (9) was adequate for this purpose. The oxidation of hydriodic acid by the substituted peroxide is slower than by hy- drogen peroxide even in the presence of molybdic acid. Except
in the lowest concentrations of the peroxide used, it was found sufficient to wait for 1 hour between the addition of the peroxide to the acid potassium iodide solution plus a few drops of molybdic acid solution and the titration with thiosulfate with starch as internal indicator.
The relationship bet.ween the amount of iodine liberated and the concentration of monoethyl hydrogen peroxide was established in the following manner. In ten flasks a 1.1 M peroxide solution was introduced in amounts varying from 0.1 to 1.0 cc. and made up to 5 cc. by addition of water. These solutions were allowed to react
1
FIQ. 1. Curve illustrating the proportionality between the amounts of ethyl hydrogen peroxide present and the amounts of 0.1 N thiosulfate required. The peroxide concentration (abscissa) is given in per cent of the maximum concentration used (0.2 M).
for 1 hour with 5 cc. of a 10 per cent potassium iodide solution plus 2 cc. of 33 per cent sulfuric acid plus 3 drops of a saturated aqueous solution of molybdic acid. The titration was carried out with 0.1 N thiosulfate solution. The determination was per- formed in duplicate. The curve in Fig. 1 was drawn through points representing the averages of the two individual values obtained. From the diagram it follows that a strictly linear re- lationship exists under the conditions of the experiment. Fur- thermore, it is to be concluded that if the reaction between the peroxide and hydriodic acid is not going to completion, the per- centage deviation from the theoretical value remains constant in
the concentration range tested. The method could therefore be used with confidence for the present purpose.
Order of Reaction-3 cc. of the undiluted Catalase XXXIV (k = 2020) and 1 cc. of M/15 phosphate buffer (pH 6.79) were placed in a 50 cc. Pyrex beaker and mechanically stirred at a moderate rate. At the experimental time = 0, 1.0 cc. of 1.1 M monoethyl hydrogen peroxide was quickly added. The reaction was stopped
TABLE II
Kinetics of Decomposition of Monoethyl Hydrogen Peroxide bv Catalase at Different Temperatures
Each titration figure given was obtained by a separate experiment.
Reac,‘;n&otpped 0.1 N thipsulfatc requned o&;og;;;$ k (monomolecular) k (mm order)
At 23”; 2 cc. Catalase XXXIV (k = 2020) + 2 cc. M/15 phosphate buffer (pH 6.79) + 1 cc. 1.1 N CzHsOOH
at various intervals by addition of 3 cc. of 33 per cent sulfuric acid. The glass stirrer was lifted above the surface of the mixture and rinsed with a few cc. of distilled water. 4 cc. of 10 per cent potassium iodide solution and 3 drops of molybdic acid solution were added. After standing for 1 hour, the liberated iodine was titrated with 0.1 N thiosulfate. Experiments were conducted at room temperature (22”) and also at 0-l’ (beaker immersed in a water-ice mixture). The solutions in the case of the latter series
were all previously cooled. The results are given in Table II. From them were calculated both the monomolecular and the zero order reaction constants, as shown in the last two columns of the table.
The calculations show that whereas the course of the reaction at the low temperature may satisfactorily be described by the equation of the first order, the data obtained at 22” yield greatly increasing monomolecular reaction constants in time and appear to fit approximately the equation of the linear relation, No ex- planation at present can be offered for this observation.
Effect of Temperature on Rate of Reaction-For these experi- ments a water thermostat was used, the temperature of which
b'1G. 2. Variation of the reaction velocity with the temperature. The: ordinate represents the amounts of substrate decomposed in 5 minutes, expressed in cc. of 0.1 N thiosulfate.
could be adjusted to any desired value between +O.l” and +99” with a constancy of f0.003”. The reaction time was 5 minutes throughout. The value for time = 0 was obtained as the aver- age of four determinations in which the sulfuric acid had been added before the substrate. The actual runs at the four tempera- tures selected were performed in quadruplicate, and the average obtained of the four values was used for the plotting of Fig. 2. From Fig. 2 it appears that there exists a linear relationship be- tween temperature and rate of reaction as expressed by turnover of substrate. &IO in the interval O-10” equals 2.3 and between lo-2o”, 2.19.
E$ect of Hydrogen Ion Concentration-In these experiments
1.0 cc. of Catalase XXXV (Ic = 5300) was added to 1.0 cc. of 1.1 M monoethyl hydrogen peroxide and 3.0 cc. of buffer mixtures ranging from pH 3.85 to 10.43. All the pH values given in this paper were obtained by measurements with the glass electrode in solutions of identical composition with that of the mixtures used for titration; these had been allowed to stand for at least 1 hour to allow the enzymatic reaction to go to completion. The author wishes to thank Mr. D. DuBois for performing the pH determina- tions with the modified glass electrode and circuit as devised by him.
The temperature in these experiments was kept near 0” by cooling with ice and water. Borate, phthalate, acetate, phos- phate, and glycocoll buffer mixtures of a molar strength of 0.06
FIG. 3. Activity-pH curve. The ordinate represents
I
the amounts of peroxide decomposed in 10 minutes, expressed in cc. of 0.1 N thiosulfate.
to 1.0 were used. No attempt was made to carry out all exieri- ments at the same ionic strength, as a variation of this value did not appear to affect the reaction rate appreciably. All experi- ments were done in duplicate. The resulting pH-activity curve is given in Fig. 3. Whereas for hydrogen peroxide as substrate catalase will show an activity optimum between pH 6.5 and 9, slightly varying according to the source of enzyme material used and to the different investigators (lo), this curve shows that the activity towards the substituted peroxide rises steadily with pH, attaining a sharp maximum near 10 and falling sharply again be- yond this value, probably due to enzyme destruction at this hy- drogen ion concentration. Control experiments showed that the rather alkaline optimum is not an artifact due to an instability of the peroxide at this pH. In spite of this finding, most of the
present experiments were conducted near pH 7 in order to exclude the possibility of any secondary reactions caused by the high hydroxyl ion concentration and in order to facilitate a comparison with the enzymatic decomposition of hydrogen peroxide which is commonly studied at a pH slightly below 7.
A$inity of Catalase for Monoethyl Hydrogen Peroxide-The so called Michaelis constant (IL), i.e. the substrate concentration at which the enzymatic reaction proceeds at half the maximal speed, was determined in a manner analogous to former experi- ments in which hydrogen peroxide was the substrate (11). The fact that monoethyl hydrogen peroxide has some affinity for
2.0
QS---------
I.0
24 2.0 1.6 4, I.2 I.4
FIQ. 4. Activity-@] curve. The abscissa represents the negative logarithm of the substrate concentration; the right ordinate, amounts of peroxide decomposed in 5 minutes, expressed in cc. of 0.1 N thiosulfate; the left ordinate, the rational measure, the maximal reaction rate being taken as 1.0.
catalase was borne out in the earlier work, when it could be shown that the inhibition of the catalase-hydrogen peroxide reaction by the substituted peroxide is of the competitive type.
Of the five series of experiments carried out with constant en- zyme concentration and varying substrate concentration, the most satisfactory one was used for plotting the curve in Fig. 4. The reaction mixtures consisted of 3.0 cc. of ~/15 phosphate buffer of pH 6.79, amounts of a 2.07 N monoethyl hydrogen peroxide solution (neutralized) varying from 0.05 to 1.0 cc., 1.0 cc. of Cata- lase XXXV (k = 5300), and distilled water to make up a total volume of 5.0 cc. The reaction time in this series was 5 minutes and the temperature near 0’. The curve shows the variation of
substrate turnover under these conditions with a variation of the total substrate concentration between 0.0207 and 0.414 N. As usual, not the absolute substrate concentration but the negative logarithm of this value is taken as the abscissa. This manner of plotting has the advantage that the experimental points corre- sponding to low substrate concentration are more evenly spaced than they would be by direct use of the concentration values. The Michaelis constant, which is the parameter of the left upward branch of the activity-PIIs] curve, is found to be nearly -log [S] = 1.4. The corresponding value for [X] is 0.04 N. The K, value for hydrogen peroxide as a substrate was formerly found to be 0.066 N = 0.033 M. Since in the case of a mono-substituted peroxide the normality equals molarity, it follows that the affinity of catalase for both substrates is very similar.
The descending branch of the activity-P[sl curve at higher sub- strate concentrations is similar to that of the curves with hydrogen peroxide as substrate (11). It is a demonstration of the fact that at high substrate concentrations the enzyme is inhibited. For ethyl hydrogen peroxide sufficient data are not yet available to determine with accuracy the value of the parameter of the de- scending branch of the activity-P[s] curve. A graphical extra- polation, however, would yield a value for this “second Michaelis constant” of the order of magnitude found for hydrogen peroxide (0.4 M).
Thermolability of Catalyst-In order to ascertain that no ther- mostable non-enzymatic factor present in the enzyme preparation is catalyzing the breakdown of the substituted peroxide, 5 cc. of Catalase XXXV (k = 5300) were pipetted in a test-tube and heated for 10 minutes in a boiling water bath. A dark brown clot formed during this procedure and was filtered off. 1 cc. of this solution when added to 3 cc. of phosphate buffer, pH 6.79, and 1 cc. of 1.1 N monoethyl hydrogen peroxide solution, did not cause a decrease of the peroxide titer within 10 minutes at 0’. In a control experiment with non-treated enzyme solution an amount of peroxide equivalent to 2 cc. of 0.1 N thiosulfate was decomposed. The catalysis is therefore due to a thermolabile agent.
Inhibition by Cyanide-In a mixture consisting of 1 cc. of 1.1 N monoethyl hydrogen peroxide, 3.9 cc. of phosphate buffer, pH 6.79, 1 cc. of Catalase XXXV (k = 5300), and 0.1 cc. of 0.1 M
sodium cyanide (neutral), no cleavage of the peroxide was ob- served within 10 minutes at 0”. The complete inhibition of the catalysis by 2 X 1O-3 M HCN suggests that a catalyst containing trivalent iron is concerned in the reaction. Catalase has been shown to contain stabilized ferric iron combined with protopor- phyrin as the prosthetic group of the enzyme (12). The reaction between catalase and hydrogen peroxide is already completely inhibited by 2 X 10e4 M HCN (11).
Reaction Products-Whereas hydrogen peroxide, when acted upon by catalase, yields molecular oxygen and water as the final reaction products, in the breakdown of monoethyl hydrogen perox- ide no appreciable amounts of gas are released. Instead, a strong odor of acetaldehyde is noticed. The formation of aldehyde was qualitatively demonstrated by the well known condensation test in the presence of strong alkali and by the blue color produced by addition of sodium nitroprusside and piperidine. It should bc mentioned, however, that even the untreated peroxide solution gave a somewhat positive aldehyde test.
In order to test for the possible formation of free acid, e.g. acetic acid, in the course of the process, 3 cc. of Catalase XXXIV (k = 2020) and 1 cc. of ~/15 phosphate buffer, pH 6.79, were placed in a small crucible. An electric stirrer, a glass electrode, and a calomel electrode were inserted in the solution. The potential value was recorded, and 1.0 cc. of 1.1 N monoethyl hydrogen peroxide solution was added. The introduction of the buffered peroxide solution caused some shift in the pH. While the reac- tion proceeded, the potentials were recorded in short intervals. There was no detectable pH change within 5 minutes; t,herefore no acid is formed in the reaction.
According to Rieche (8), monoethyl hydrogen peroxide may break down in various ways, depending on the agent used; e.g., on attack by alkali at higher temperatures only very little gas is formed, whereas under the action of formaldehyde and alkali much hydrogen and, moreover, formic acid, acetic acid, ethyl alcohol, and acetaldehyde are formed in a vigorous reaction.
It is planned to subject the enzymatic breakdown of the perox- ide to further and quantitative analysis.
Optical Study of Intermediate Enzyme-Substrate Compound. Technique-The exact position of the absorption bands of the in- termediate was determined with a Hilger wave-length spectrom-
eter. The accuracy of the setting of the wave-length drum was checked by the emission lines of an electrical sodium burner (Zeiss). For the other observations calibrated pocket spectro- scopes (Brown, Zeiss) were used. Such straight vision instru- ments of small dispersion permit a better definition of absorption bands and an easier spotting of weak bands than big spectroscopes of the Kirchhoff-Bunsen type. Most of the observations were made visually, but the whole spectral cycle was also photographi- cally recorded by means of a miniature roll film camera of wide
FIG. 5. Schematic representation of the arrangement used for optical study of the enzyme-substrate compound: A represents the projection lantern; B, condenser; C, trough for cooling; D, experimental vessel; E, motor and stirrer; F, pocket spectroscope; G, roll film camera.
aperture (f = 2.9) and supersensitive panchromatic film (Eastman).
In those experiments where rapid mixing of enzyme and sub- strate was desired, a Pyrex tubularly-shaped cell with fused on windows and an inserted glass stirrer was used. The enzyme, buffer, and substrate solutions were stratified above each other by means of different concentrations of sucrose and the motor stirrer started at 0 time as suggested by Stadie (13). It was as- certained that sucrose in the concentrations used is no inhibitor
for the enzyme reaction. The details of the arrangement are shown in Fig. 5 which is largely self-explanatory.
Qualitative Observations-On direct visual observation in trans- mitted light and in the thickness of layer used for the spectro- scopic experiments the enzyme solutions appear brown in color. Upon the addition of monoethyl hydrogen peroxide, there is a rapid color change to a greenish hue. Within the following
6fO 690 5$0 590 +fomp
6tO 6bO a-60 a-60 &+.
FIG. 6. Schematic representation of the spectroscopic cycle. Z repre- sents the spectrum of free enzyme; II, lag period during which a greenish color but no discrete absorption bands are noticed; ZZZ, spectrum of en- zyme-substrate compound; IV, coexisting intermediate and free enzyme; V, restored enzyme spectrum. (After direct observation with the spectroscope. The heights of the bands indicate their visual intensity.)
seconds the red color of the intermediate catalase-peroxide com- pound appears. In the course of the breakdown of the compound which requires time of the order of minutes, the red tint fades and with the reformation of the free enzyme the original brown color is restored. The corresponding changes in light absorption, as observed with a small spectroscope, are represented schemati- cally in Fig. 6. No specific absorption bands are visible during
the greenish transition period (II). It is therefore possible that this color shade is due neither to the enzyme nor to an intermediate but to concomitant pigments in the enzyme solutions (biliverdin, hepatoflavin), the absorption bands of which are located in the far red and violet regions respectively.
The whole cyclic phenomenon may again be released by ad- dition of fresh substrate. The restoration of the original enzyme spectrum is accompanied by the disappearance of titratable perox- ide from the system.
Position of Absorption Bands of Intermediate-In order to pro- long the visibility of the spectrum of the enzyme-substrate com- plex, the measurement was carried out at 0”. 5 cc. of Catalase XXIX (k = 7125) and 5 cc. of a 0.41 N monoethyl hydrogen peroxide were cooled separately in ice. A small absorption tube of the Baly type with fused on windows and ground joints (Schott and Genossen, Jena) was also cooled. The solutions were mixed and transferred at once to the tube. The tube was submersed in an ice-filled trough. In the Hilger spectrometer, the long wave absorption band of the intermediate was better defined and more intense than the short wave band. Below are given the data obtained together with the values reported by Zeile and Hcll- Strom (4) for the uncombined enzyme, with which values our own observations were in satisfactory agreement.
Spectrum of Free Enzyme-
I. 650. (646-620. ,610; II. 550-530...520...510-490 mp
-&i- -z- -
500
Spectrum of Enzyme-Substrate Compound-
I. 576-564; II. 540-529 mp
-zi- -
534.5
E$ect of Temperature on Rate of Formation of Intermediate-- The arrangement shown in Fig. 5 was used, with the exception of the film camera. The vessel with the electric stirrer in place was dipped into the trough filled with water of room temperature (22’). The bottom layer in the experimental vessel consisted of 15 cc. of a mixture of 10 parts of Catalase XXXVI (k = 4525) and 5 parts of a sucrose solution prepared by dissolving 100 gm.
of pure sucrose in 100 cc. of water. A layer of 2.5 cc. of a mixture of 20 parts of ~/15 phosphate buffer (pH 6.79) and 5 parts of the sucrose solution was carefully stratified above the bottom layer. The top or third layer was formed by 2.5 cc. of 2.07 N
monoethyl hydrogen peroxide solution containing no sucrose. The electric stirrer was started at 0 time, and the interval lapsing before the stronger absorption band of the enzyme-substrate compound at 570 mp could be first detected by visual observation through the pocket spectroscope was recorded. For three runs the time intervals 4.5, 3.6, and 3.4 seconds were obtained, averag- ing 3.8 seconds. In the following series the side compartments of the external trough were filled with ice. They were separated from the middle compartment which contained water, by means of metal gauze. Before beginning the experiment the vessel con- taining the three layers of solution was cooled for at least 15 minutes. The temperature of the ice water surrounding the ves- sel was 2”. Under these conditions the time intervals recorded in three trials were 6.3,6.4, and 6.3 seconds, averaging 6.3 seconds. It follows that from 2-22’ the rate of formation of the intermc- diate increases 1.6 times. Assuming a linear relationship as in the case of the over-all reaction, Q10 in this interval would be 2/1.6 = 1.26. This value is smaller than Q~o for the over-all reaction (Table III). It is preferable not to base calculations of the apparent critical increment of the combination of the enzyme with the substrate on this result which was obtained with a primi- tive technique. It is probable, however, that the apparent ac- tivation energy of this reaction is somewhat smaller than that of the over-all reaction, and more specifically of the breakdown of the enzyme-substrate compound.
Effect of Hydrogen Ion Concentration-With the external trough filled with water of room temperature (22”), similar experiments were conducted with buffers of different pH. It was found that variation of pH between 4.11 and 8.82 is without a significant effect on the rate of the formation of the enzyme-substrate com- pound. Nine readings at four different hydrogen ion concentra- tions yielded an average time value of 4.0 seconds, the highest figure obtained being 5.3 and the lowest 3.4 seconds.
The pH values given were obtained by measurements with the glass electrode after the experiment.
Experiments on Reality of Observed Lag Period-The time neces- sary for complete mixing under the conditions of the preceding experiments was determined by measuring the length of time re- quired for uniform distribution throughout the system of a top layer containing methylene blue dissolved in water. The bottom layer consisted of 10 cc. of water and 5 cc. of sucrose solution; the middle layer contained 2.5 cc. of a mixture of 20 parts of water with 5 parts of sucrose solution. Direct visual observation yielded a time value of 1.6 and 1.4 seconds, respectively. If the spectroscope,was used, values of 2 seconds were obtained in two experiments. It follows that although the time necessary for complete mixing was comparatively long, the above rates observed for the appearance of the intermediate were distinctly slower than would be warranted by the mixing time. However, it ap- peared desirable to decrease the mixing time considerably so as to make it negligible compared with the observed time lag in the formation of the intermediate. Dr. W. C. Stadie, who had used a similar arrangement for the study of carbonic anhydrase (13), was good enough to advise the author on this aspect. Conse- quently, the rate of the stirring device was increased. Further- more, the specific gravity of the different layers was lowered by the use of more dilute sucrose solutions. In order to avoid the foaming which occurred at the great speed of stirring, it was necessary but also sufficient to connect the motor with a Morse telegraph switch and to press the switch down for the length of time necessary to start the stop-watch. The sucrose content of the bottom layer in the final experiments was 5 per cent, in the middle layer 2.5 per cent, and in the top layer 0. By means of methylene blue control experiments, it was found that a stirring time of 0.2 to 0.3 second was sufficient to obtain uniform mixing. In three runs with the enzyme and substrate mixture at pH 6.6 and 22’ the time lapsing before the first appearance of the stronger absorption band of the intermediate was found to be 1.5, 2.3, and 2.0 seconds, respectively. Since in these last experiments the mixing time was small compared with the observed lag period, some confidence may be placed in the reality of its existence.
Amount of Substrate Required for Complete Transformation into Intermediate--It had been observed in preliminary experiments that if a small amount of monoethyl hydrogen peroxide was added
to a highly active catalase solution, the absorption band of the enzyme with the center at 629 mp was not completely extinguished but that a certain residual absorption in this region remained while the absorption bands of the enzyme-substrate compound ap- peared. The amount of substrate required for complete trans- formation into the intermediate was determined by adding the substrate solution from a burette to the enzyme solution at low temperature under spectroscopic observation. The titration has to be carried out rapidly, as the breakdown of the intermediate also yields amounts of free enzyme increasing with time.
2.0 cc. of Catalase XXIX (k = 7125) required 2.95 and 3.0 cc. of 0.94 N monoethyl hydrogen peroxide (neutralized with phosphate buffer) for the complete temporary abolition of the enzyme absorption band in the red. In a later experiment, 15 cc. of Catalase XXXVI (k = 4525) required 6.55 cc. of a 2.07 N peroxide solution. Both titrations were performed at 3-4’.
The number of enzyme molecules present may be computed by using the result of Haldane’s (14) calculations, based on Zeile’s (4) experimental data, according to which 1 molecule of liver catalase will decompose at 0” and at a substrate concentration of 0.01 M, 5.4 X lo4 molecules of hydrogen peroxide per second. On Haldane’s assumption that 1 enzyme molecule contains 1 iron atom and therefore one active center and with the molecular weight of catalase taken to be 68,000, as found by measuring the rate of diffusion of the enzyme (Stern (15)), it can readily be shown that under the conditions of the assay 1.26 mg. of enzyme will decompose 1 mM of hydrogen peroxide per second. On this basis Catalase XXIX (k = 7125) contained 0.214 mg. of enzyme per cc. or 3 X 1OW mM of catalase. In the titration experiment cited above, 2 cc. of this enzyme solution or 6 X 10h6 mM of cat- alase required 3 cc. of 1 N monoethyl hydrogen peroxide or 3 mM of the substrate for the optical end-point. Therefore 1 en- zyme molecule requires 5 X lo5 substrate molecules for the com- plete transformation into the intermediate. From the titration experiment with Catalase XXXVI (see above) a very similar figure may be obtained.
Obviously this ratio does not imply necessarily that 1 mole- cule of the enzyme-substrate compound consists of 1 catalase molecule and 5 X lo5 peroxide molecules. It simply means that
an excess of substrate of this order is required to shift the equilib- rium in the reaction
Enzyme + substrate e enzyme-substrate compound
entirely to the right. In other words, the probability that each enzyme molecule, whether originally uncombined or whether re- leased on the breakdown of the intermediate, will recombine im- mediately with fresh substrate molecules, approaches unity. The figure is therefore rather of statistical than of a stoichiometrical character. In particular, it does not indicate how many sub- strate molecules will combine simultaneously with 1 enzyme molecule.
The figure given may require a correction for two reasons. The lag period mentioned above has not been taken into oonsideration in the titration experiment. This would tend to make the ratio appear too large. On the other hand, if 1 enzyme molecule does not contain 1 but 4 iron atoms, as does hemoglobin, then the ratio given would have to be multiplied by 4.
Formation of Intermediate While Enzyme Is Adsorbed-As has been reported in a recent note (16), the spectrum of the enzyme remains unchanged while it is adsorbed on aluminum hydroxide gel or on silica gel, On addition of monoethyl hydrogen peroxide to the adsorbate in suspension, the peroxide is decomposed and the spectrum of the intermediate is observed as with freely dissolved enzyme. It follows that the combination of the enzyme with the substrate takes place at a grouping of the catalase molecule which is different from that which is attached to the adsorbent. Since the spectrum of the enzyme is not altered by the adsorption but is changed by the addition of substrate, the conclusion appears to be warranted that combination of enzyme and adsorbent takes place by means of the protein carrier of the enzyme, while the substrate combines with the hemin group of the enzyme, causing the specific light absorption in the visible range of the spectrum.
DISCUSSION
Speci$city of Catalase-Catalase has been considered to be the prototype of an enzyme exhibiting an absolute specificity. Here- tofore the only substrate known to be attacked by this enzyme was hydrogen peroxide. It has been maintained that neither
alkylated peroxides (17) nor substances of the type of perbenzoic or peracetic acid (18) are affected by catalase. This seemed to provide a significant contrast to peroxidase which in the presence of suitable oxygen acceptors may not only utilize hydrogen perox- ide but also ethyl hydrogen peroxide and, to a lesser degree, per- acetic acid (19, 20). That monoethyl hydrogen peroxide shows some affinity for catalase was demonstrated several years ago (11). It was then found that the addition of this compound to a system containing catalase and hydrogen peroxide will cause an inhibition of the decomposition of the latter. By varying the hydrogen peroxide concentration alone, it was shown that the inhibition is decreased by increasing the hydrogen peroxide con- centration. It was therefore concluded that the inhibitory effect of ethyl hydrogen peroxide is of the competitive type. In those experiments, as in the work of the other authors, highly diluted catalase preparations were used. It was only when the sub- stituted peroxide was added to very concentrated enzyme solutions that the decomposition of this substrate and the formation of a spectroscopically visible intermediate were discovered (3). This fact is proof that the specificity of catalase is of a relative and not of an absolute character. Hydrogen peroxide appears to repre- sent the most readily attacked substrate, while the monoethyl hydrogen peroxide is affected at a much smaller rate. However, as the present study shows, the decomposition of this compound by catalase may be studied in a manner similar to the cleavage of hydrogen peroxide provided sufficiently active enzyme prepara- tions are used.
The physiological function of catalase has hitherto been dis- cussed in a purely speculative manner, inasmuch as all attempts to demonstrate the occurrence of its supposedly unique substrate, hydrogen peroxide, in the cells of higher animals have failed. The demonstration that a substituted organic peroxide may serve as a substrate of this enzyme together with the fact that sufficiently high catalase concentrations to effect such a catalysis have been found to exist in the liver of mammals suggests a new approach to the problem.
Comparison between Hydrogen Peroxide and Ethyl Hydrogen Peroxide As Substrates of Catalase-Two points of major interest emerge from the comparative study of the action of liver catalase on these two substrates. Even at low temperatures it has not
yet been possible to observe the appearance of the spectrum of an intermediate when hydrogen peroxide is decomposed. The only explanation suggested at present is a greater rate of break- down of the enzyme-substrate compound in this case (see Table III) .2 On decomposition of hydrogen peroxide molecular oxygen and water are the reaction products. Monoethyl hydrogen perox- ide, on the other hand, yields no gaseous products on enzymatic cleavage but acetaldehyde and other unidentified compounds. The aldehyde formation from a simple organic peroxide is ob- viously of physiological interest.
TABLE III
Catalysis of Hydrogen Peroxide and Ethyl Hydrogen Peroxide by Liver Catalase (Over-All Reaction)
The data for hydrogen peroxide as substrate were taken from the litera- ture (cf. Haldane and Stern (IO); Stern (11)).
-
Kinetics of over-all reaction at 0”. \/lonomolecuIa ,‘ t, I‘ ,‘ “ 20”.... I<
pH, activity optimum . . . . _. 1st Michaelis constant (Km), M., 2nd (‘ LL “ I‘, Temperature coefficient, Q,.. _, No. of substrate molecules destroyed by
1 enzyme molecule at O”, pH 6.6, total substrate concentration 0.62 N..
6.5-O 0 ,033 0.4 1.4 (O-20”)
5.4 x 104
Hydrogen pcroxid IS--O-O-H
- 0
-
r *
-
Ethyl hydrogen peroxide
C2Ha--O--O~--H
Monomolecular 0 order
10 0.04
Same order 2.2 (0620”)
1.2 x IO”1
*The velocity constants are decreasing with time, owing to enzyme inactivation by the substrate.
t Calculated from the determination incorporated in the curve of Fig. 4.
In Table III are compiled some features of the catalysis of both substrates by liver catalase.
Comparison between Catalase and Methemoglobin As Catalysts-
2 In order to observe the spectrum of the intermediate compound the enzyme must be kept saturated with substrate. Since 1 molecule of cata- lasa, if saturated with substrate, splits 2 X lo5 molecules of H&z per second at 0” (Haldane (14)), for a catalase preparation of k = 7000 a HsOs concen- tration of 6 M would be required for an observation time of 10 seconds. This is experimentally not feasible because the reaction will proceed at an explosive rate and because the enzyme is quickly destroyed by high Hz02 concentrations.
The methemoglobin catalysis of hydrogen peroxide and ethyl hy- drogen peroxide is more than a model reaction. Catalase and methemoglobin have an identical prosthetic group, namely para- hematin (12). The much smaller catalytic efficiency of methe- moglobin is due to the difference in the nature of the protein car- rier. Methemoglobin will only decompose of the order of 1OV hydrogen peroxide molecules per catalyst molecule per second (21). Figures for the ethyl hydrogen peroxide catalysis are not yet available. In contrast to catalase, methemoglobin will form intermediate compounds of a distinct absorption spectrum with both substrates. The pattern of the spectra of the two unstable complexes resembles closely that of the catalase-ethyl hydrogen peroxide compound. The complex of methemoglobin with hy- drogen peroxide was discovered 36 years ago by Kobert (22). Its light absorption and composition were recently studied by Hauro- witz (23). Haurowitz concludes that the molecular ratio in this case is unity and that the complex contains ferric iron to which the peroxide is linked by coordinative valencies. The complex of methemoglobin with ethyl hydrogen peroxide was recently dc- scribed by the author (3) and independently by Keilin and Har- tree (24). An excess of only 8 peroxide molecules per methemo- globin molecule is sufficient to suppress the absorption band of methemoglobin in the red, compared with an excess of the order of lo6 molecules in the case of catalase. The ratio of these figures resembles that of the catalytic activity of the enzyme and of the blood pigment.
On Mechanism of Enzyme Reaction-The present study sheds some light on the mechanism of an enzyme action. The catalyst operates by providing a new path of reaction which leads over an intermediate composed of enzyme and substrate molecules. This compound is unstable but has a mean span of life sufficient to allow for direct observation. It should be emphasized that the interpretation of the observations reported in the present paper is consistent with but not dependent on the validity of the evidence offered for the constitution of the enzyme (4, 12). It is felt that the interpretation is justified by the agreement of the data ob- tained by the optical and volumetric methods employed. The time required by the spectral cycle to go to completion equals the time required for complete decomposition of the substrate.
The close analogy with the non-enzymatic methemoglobin catalysis suggests a similar constitution of the intermediate com- pounds. In agreement with the interpretation by Haurowitz (23) of the methemoglobin-hydrogen peroxide complex, the cat- alase-ethyl hydrogen peroxide complex may be depicted as a covalency compound, where t,he peroxide molecule is linked to the coordinately tetravalent ferric iron of the hematin group of the enzyme. It appears probable that a similar intermediate occurs during the catalase-hydrogen peroxide catalysis, though hitherto attempts to demonstrate its formation have failed.
It is shown that the rate of formation of the enzyme-substrate compound is rapid compared with the rate of the over-all reac- tion. The kinetics of the latter are therefore governed by other steps in the series of reactions. The effect of hydrogen ion con- centration and of temperature as observed in the study of the total process obviously concerns the later reaction phases. It is quite possible that the actual course of events is more complex than the spectroscopic findings would indicate. The product molecules formed by the breakdown of the intermediate may be radicals, initiating a chain reaction in which the original catalyst no longer participates (11, 25). The dependence of the rate of the over-all reaction on temperature indicates that at any given instant only a fraction of the molecules of the intermediate is in an activated state.
It remains to be seen to which extent the findings of this study apply bo enzyme action in general.
SUMMARY
1. A study was made of the decomposition of monoethyl hy- drogen peroxide by liver catalase. A volumetric procedure was used for the assay of the peroxide. In this manner, the kinetics and the effect of temperature, of pH, of varying the substrate concentration, and of cyanide on the enzyme reaction were studied. The results are compared with those obtained with hydrogen peroxide as substrate.
2. In the course of the enzymatic process there is formed an intermediate compound with a characteristic absorption spectrum. The intermediate is unstable; it breaks down to form free enzyme and reaction products. It exhibits the properties postulated by
Michaelis and Menten for an enzyme-substrate compound. A preliminary optical study of this compound has revealed that it is not a mere adsorption complex; that the rate of formation of the enzyme-substrate compound is great compared with that of the total reaction; that it has a smaller temperature coefficient than the over-all reaction; that it is independent of pH between 4 and 9.
BIBLIOGRAPHY
1. Hem-i, V., Lois g&kales de l’action des diastases, Paris (1903). 2. Michaelis, L., and Menten, M. L., Biochem. Z., 49,333 (1913). 3. Stern, K. G., Nature, 136, 335 (1935). 4. Zeile, K., and HellsMm, H., 2. physiol. Chem., 192, 171 (1930). 5. Baeyer, A., and Villiger, V., Ber. them. Ges., 34,738 (1901). 6. Rieche, A., Ber. them. Ges., 62, 218 (1929). 7. Rieche, A., and Hitz, F., Ber. them. Ges., 62, 2473 (1929). 8. Rieche, A., Alkylperoxyde und Ozonide, Dresden, 23 (1931). 9. Stern, K. G., Z. physiol. Chem., 204, 259 (1932).
10. Haldane, J. B. S., and Stern, K. G., Allgemeine Chemie der Enzyme, Dresden (1932).
11. Stern, K. G., Z. physiol. Chem., 209, 176 (1932). 12. Stern, K. G., J. Biol. Chem., 112,661 (1936). 13. Stadie, W. C., and O’Brien, H., J. Biol. Chem., 103,521 (1933). 14. Haldane, J. B. S., Proc. Roy. Sot. London, Series B, 106, 559 (1931). 15. Stern, K. G., Z. physiol. Chem., 217, 237 (1933). 16. Stern, K. G., Science, 63, 190 (1936). 17. Bach, A., and Chodat, R., Ber. them. Ges., 36, 1756 (1903). 18. Freer, P. C., and Novy, F. G., J. Am. Chem. Sot., 27, 161 (1902). 19. Grimmer, W., MiZchwirtschuJt. Zentr., 44, 246 (1915). 20. Wieland, H., and Sutter, H., Ber. them. Ges., 63,73 (1930). 21. Stern, K. G., Z. physiol. Chem., 216,35 (1933). 22. Kobert, R., Arch. ges. Physiol., 82,603 (1900). 23. Haurowitz, F., Z. physiol. Chem., 232, 159 (1935). 24. Keilin, D., and Hartree, E. F., Proc. Roy. Sot. London, Series B, 117,
1 (1935). 25. Haber, F., and Willst~itter, R., Ber. them. Ges., 64, 2844 (1931).
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