Differences in Activity and Reaction of Bacterial and Erythrocyte Catalase with Peroxides, Study notes of Chemistry

Download Differences in Activity and Reaction of Bacterial and Erythrocyte Catalase with Peroxides and more Study notes Chemistry in PDF only on Docsity! 402 B. CHANCE I950 catalase haematins are bound as complex II and at pH 3*6 nearly all are, under the particular experi- mental conditions. 8. The formation of complex II is responsible for the decrease of catalase activity during the usual Kat. f. determination and for the rapid decrease of catalase activity in acid solutions. 9. The steady-state concentration of hydrogen peroxide in the presence of catalase and the notatin system is very small. Under particular experimental conditions, the hydrogen peroxide concentration is calculated to be of the order of 10-9M. 10. The very rapid reaction of catalase-bound methyl hydrogen peroxide I with hydrogen peroxide can be studied m greater detail when hydrogen per- oxide is continuously generated by the notatin system. The velocity constant for this reaction is of the same order as that ofcatalase-hydrogen peroxide I with hydrogen peroxide (3.5 x.107M-1 sec.-'). In this reaction, hydrogen peroxide acts as an acceptor as in peroxidatic reactions of complex I with ethanol, but is about 30,000 times as active as ethanol. The author owes many thanks to Prof. D. Keilin, F.R.S., and to Dr E. F. Hartree for their helpful suggestions in the course of this work and on the content of this paper. He is also grateful for the facilities granted him at the Molteno .Institute and for supplies of purified notatin. REFERENCES Bonnichsen, R. K. (1948). Dissertation, Karolinska Insti- tutets, Stockholm. Bonnichsen, R. K., Chance, B. & Theorell, H. (1947). Acta chem. 8cand. 1, 685. Chance, B. (1947a). Acta chem. 8cand. 1, 236. Chance, B. (1947b). Rev. Sci. Instrum. 18, 601. Chance, B. (1948). Nature, Lond., 161, 914. Chance, B. (1949a). J. biol. Chem. 179, 1299. Chance, B. (1949b). J. biol. Chem. 179, 1311. Chance, B. (1949c). J. biol. Chem. 179, 1331. Chance, B. (1949d). J. biol. Chem. 179, 1341. Chance, B. (1949e). J. biol. Chem. 180, 947. Davies, P. W. & Brink, F. (1942). Rev. Sci. In8trum. 13, 524. Euler, H. von & Josephson, K. (1927). Leibig8 Ann. 4-152, 158. George, P. (1947). Nature, Lond., 160, 41. George, P. (1948). Biochem. J. 43, 287. George, P. (1949). Biochem. J. 44, 197. Keilin, D. & Hartree, E. F. (1936). Proc. roy. Soc. B, 119, 141. Keilin, D. & Hartree, E. F. (1945). Biochem. J. 39, 293. Keilin, D. & Hartree, E. F. (1948). Biochem. J. 42, 221. Lemberg, R. & Foulkes, E. C. (1948). Nature, Lond., 161, 131. Lineweaver, H. & Burke, D. (1934). J. Amer. chem. Soc. 56, 658. Millikan, G. A. (1936). Proc. roy. Soc. A, 155, 277. Roughton, F. J. W. & Millikan, G. A. (1936). Proc. roy. Soc. A, 155, 258. Stead, B., Page, F. M. &]Denbigh, K. G. (1948). Trans. Faraday Soc. 44, 479. Theorell, H. (1941). Enzymologia, 10, 250. The Enzyme-substrate Compounds of Bacterial Catalase and Peroxides* BY B. CHANCE AND D. HERBERT The Johnson Re8earch Foundation, Univer8ity of Pennsylvania, U.S.A., and Medical Research Council Unit for Bacterial Chemistry, Lister Institute, London S.W. 1 (Received 5 September 1949) One of the approaches to the relation between en- zyme activity and the nature of the protein com- ponent is the study of enzymes with dissimilar pro- tein components and identical prosthetic groups. A comparative study of the peroxidatic activity of horse radish peroxidase and ferrimyoglobin affords a gross example ofthe potent influence ofthe protein component. But here the effect is too great; ferri- myoglobinispracticallycompletely inactive. Amore sensitive investigation ofthe differences between the activities of erythrocyte catalase and the recently crystallized bacterial catalase (Herbert & Pinsent, 1948) would seem to be more fruitful, and is the subject of this paper. Both erythrocyte and bacterial catalases ap- parently have four protohaematin prosthetic groups attached to protein molecules of very nearly the same molecular weight. However, Herbert & Pinsent (1948) obtained definite evidence that differences exist between the two protein com- ponents, especially as indicated by their relative stability in organic solvents and at low values ofpH. There is also good evidence that the activity of bacterial catalase is different from that of erythro-* Paper No. 14 of a series on Catalases and Peroxidases. COMPOUNDS OF BACTERIAL CATALASE AND PEROXIDES cyte catalase (the values of 'Katalasefahigkeit' (Kat. f.) found by the above authors were 95,000 and 61,000 respectively). Thus these two catalases are suitable for a quantitative study of the effect of the protein component upon the activity of the two enzymes. This paper gives detailed data on the spectro- scopic and kinetic properties of the compounds of bacterial catalase with hydrogen peroxide and with alkyl hydrogen peroxides. The data show that the mechanism of action of this enzyme is identical with that of erythrocyte catalase. However, the reaction velocity constants for the combination of bacterial catalase with peroxides and for the reactions of the bacterial catalase-peroxide complexes with alcohols and related substances show significant differences from those oferythrocyte catalase; bacterial catalase reacts more rapidly with small molecules and more slowly with larger molecules than erythrocyte cata- lase. The difference between the activities of these two enzymesmaythus be attributed to a difference of protein structure. It is suggested that the haematins of bacterial catalase are more deeply 'buried' in the protein molecule than are those of erythrocyte catalase, and reactions with larger molecules are thus impeded. But the activity of the haematins of bacterial catalase towards the small hydrogen per- oxide molecule is enhanced by the proximity of the protein component and the prosthetic groups. EXPERIMENTAL Light absorption measurements. Unless otherwise stated, a Beckman quartz spectrophotometer was used, and all results are given in terms of the millimolecular extinction coefficient Em.=E/cl (L.mmol.-l cm.-'), where E is the extinction (log Is/I), c the mm-concentration and I the optical depth in cm. Wavelengths (in m,u.) are indicated by superscripts, e.g. EM. In the case of catalase, c is expressed in the units mol. catalase/l. and not mol. catalase-haematin!l. as employed by some workers. Preparations. Bacterial catalase was prepared from Micrococcus lysodeikticus by the method previously de- scribed (Herbert & Pinsent, 1948). To conserve material, the preparations were not carried through to crystallization, and represented the material obtained after stage 7 of the above procedure; they were only about 60-70% pure, but were free from other haematin compounds. Enzyme concentrations. The content of pure catalase in solutions of the approx. 60% pure enzyme was determined by (a) haematin determinations by the pyridine haemo- chromogen method, (b) spectrophotometric measurement of the Soret absorption band of catalase at 405 m,u. (a) Pyridine haemochromogen was determined by spec- trophotometric measurements at two wavelengths, the peak of the a absorption band and the minlmum between the a and P bands. According to recent measurements by Biorck (1949) with the Beckman spectrophotometer, these lie at 556 and 540 mp. respectively, and the difference between the molecular extinction coefficients is (e556 - E540) =220 x 103. Hence, when measurements are made in an optical depth of 1 cm., Haematin Fe concentration (mM) = (E556 - E540)/22.0. These values are slightly different from those obtained by Drabkin (1941), using a different type of instrument; since our measurements were made with a Beckman spectro- photometer, Bi6rck's data were used. (b) -The millimolecular extinction coefficient of bacterial catalase at the peak of the Soret band was obtained by measuring the absorption at 405 mp. of solutions stand- ardized by method (a), and is e405=405 ±5. This value was thereafter used for routine determinations of catalase con- centration. It is 6-7% higher than the corresponding values for erythrocyte catalase given by Bonnichsen (1949), and Bonnichsen, Chance & Theorell (1947). For these bacterial catalase preparations, the value of e2l0 is 15 to 2 times that of 6405 RESULTS Activity towards hydrogen peroxide In previous work on bacterial catalase the activity was expressed as Kat. f., and measured by the titra- tion method of Sumner (1941), using catalase con- centrations of approximately 0-006j,g./ml. which gave a halftime for the reaction of about 10 min. By this method, the value found for Kat. f. was 95,000 (Herbert & Pinsent, 1948). Bonnichsen et al. (1947) pointed out the disadvantages of this technique, the main one being that considerable destruction of enzyme by the hydrogen peroxide occurs, giving falling values ofthe velocity constant k1, which must be extrapolated to zero time to obtain the true value, an unsatisfactory procedure. The activity of bacterial catalase was therefore redetermined by the rapid titration method of the above authors, using considerably higher enzyme concentrations (approx. 0-12 ,ug./ml.) which gave a halftime of approx. 30 sec. Under these conditions, no appreciable destruction of enzyme occurred and the value of k remained satisfactorily constant throughout the reaction. Undersuch conditions, the destruction ofhydrogen peroxide by catalase follows exactly the equation (Bonnichsen et al. 1947) dtXkex (la) and kl is calculated from the titration results as kl= -ln-, et Xt (1 b) where e and x are the molar concentrations of cata- lase and hydrogen peroxide, and t is the time in sec. Kat. f. is related to k' by the equation: 520 kclKat. f.= M ' (Ilc) where M is the molecular weight of bacterial cata- lase (230,000). VoI. 46 403 B. CHANCE AND D. HERBERT shown by the curves I and II of Fig. 4. The spectrum of the secondary complex is readily measured in the ordinary spectrophotometer upon the addition of an excess ofmethyl hydrogen peroxide (approx. 200 ltM) to bacterial catalase (approx. 0-5 pM). The primary complex is, however, unstable, and either spon- taneously decomposes into free catalase or is con- verted into the secondary complex in a short time. The spectrum of complex I is therefore measured in the rapid-flow apparatus. The changes of extinction coefficient from free catalase to complex I and from complex I to complex II are measured according to the method described previously (Chance, 1949 c). In the region 370-420 m,u., the extinction coefficients basedupon the conversion ofcomplex Ito complex II are considered the more accurate, while those based upon the conversion of catalase to complex I are consideredmore accurate inthe region above 420m,u. In this way the spectrum of Fig. 4 is constructed. It was noted that the values ofthe change ofextinction coefficient from free catalase to complex I in the region 380-410 mu. lie somewhat above the curve based upon the change from complex I to complex II given in Fig. 4. This discrepancy was observed pre- viously (Chance, 1949c), and is again attributed to the large spectral interval required of the mono- chromator employed with the rapid-flow apparatus. The composition of the bacterial catala8e-hydrogen peroxide complex: 8pectrophotometric data The change of eg from free catalase to the primary methyl hydrogen peroxide complex (200) is much larger than that shown in Fig. 3 for the hydrogen peroxide complex (82). Since this change is identical for the primary peroxidase hydrogen peroxide and methyl hydrogen peroxide complexes (Chance, 1949g), it is here assumed thatthe change of extinction coefficient from the free enzyme to the primary peroxide complex is the same per haematin group for hydrogen peroxide and methyl hydrogen peroxide. Thus the smaller value for the bacterial catalase-hydrogen peroxide complex may be attri- buted to the fact that not all the catalase haematins are bound to hydrogen peroxide. Quantitatively, only 82/200 = 1-6 of the four catalase haematins are so bound. A similar effect obtains in erythrocyte catalases, but here 1-0 + 0- 1 haematins are bound according to studies by a similar method (Chance, 1950 a). Thus there is a higher steady-state concentration of the catalase-hydrogen peroxide complex in the reaction of bacterial catalase than in the reaction of ery- throcyte catalase with hydrogen peroxide. A graphic illustration of the difference of the extinction coefficients of the hydrogen peroxide and methyl hydrogen peroxide complexes is given in Fig. 5. The cuvette (d= 1 cm.) ofa recording spectro- photometer is initially filled with a dilute solution of bacterial catalase. As a control the solution is initially stirred, and, except for the momentary increase of optical density caused by stirring, the base-line is unaffected. Upon addition of hydrogen peroxide to a concentration of 67 ztM, an abrupt decrease of extinction is recorded and is caused by the formation of the primary catalase- hydrogen peroxide complex. The addition of hydro- gen peroxide is repeated and causes no further de- crease of extinction. This indicates that the satura- tion value ofthe catalase-hydrogen peroxide complex 15 sec. k- 008pzM-bacterial A/ catalase Bacterial catalase hydrogen peroxide Bacterial catalase methyl hydrogen @ peroxide Stirred 67Mm- 67IsM- 67,LM- 671tM- H202 H202 CH300H CH300H Fig. 5. Spectrophotometric recording of the reactions of bacterial catalase with hydrogen peroxide and with methyl hydrogen peroxide. Modified Beckman spectro- photometer, 1 cm. cuvette; 00lM-phosphate, pH 7 0; A=405 mp. has been measured. The next addition is of methyl hydrogen peroxide which combines with all the cata- lase haematins and thereby causes a large decrease of extinction. Further addition of methyl hydrogen peroxide causes no increase in the concentration of catalase-methyl hydrogen peroxide. Since satura- tion with peroxide has been achieved in these two cases, and since it is assumed that the extinction co- efficients of the two peroxide compounds are the same on a haematin-iron basis, the fraction of cata- lase haematins bound in the hydrogen peroxide complex is 7-5/20.5 scale divisions of Fig. 5. For a four-haematin catalase this corresponds to 1-5 haematins bound. This is in fair agreement with the value of 1-6 measured in Fig. 3. Cyanide results The composition of the mammalian catalase- hydrogen peroxide complexes has been determined by measuring spectrophotometrically the relative amounts of catalase-cyanide formed by the free catalase or by the catalase-hydrogen peroxide com- plex (Chance, 1949 b). In those tests, conditions were specified under which a successful experiment could be performed. These conditions involved the dissociation constant of catalase-cyanide. For this reason, the properties of bacterial catalase-cyanide are given. The spectrum of bacterial catalase- cyanide in the region 370-460 m,. is shown in Fig. 6, and is seen to be very similar to that of erythrocyte 406 I950 COMPOUNDS OF BACTERIAL CATALASE AND PEROXIDES catalase-cyanide (Chance, 1949 c). = is 360 and is 13 % greater than the corresponding value for ery- throcyte catalase-cyanide. The titration of bacterial catalase with cyanide is illustrated in Fig. 7, and the values of the apparent dissociation constant are computed by the usual formula. The average is 21-8 x 10-6M. This is over five times the value found for the equilibrium con- stant of erythrocyte catalase and cyanide (Chance, 1949a). This change is apparently not caused by a difference in the rates of formation of the cyanide compounds; measurements ofthis value for bacterial catalase give about 2 x 106M-1 sec.-' compared with 0-9 x 106 for mammalian catalases (Chance, 1949a). r200 0 I0 370 400 430 460 Wavelength (mu.) Fig. 6. The spectrum of bacterial catalase-cyanide in the region of the Soret band (Curve B). Final concentra- tions: 0-4pi-bacterial catalase, 400 pLM-hydrocyanic acid, 001m-phosphate, pE 7 0. Curve A, free bacterial catalase for comparison. Thus it is the velocity constant for the dissociation of bacterial catalase-cyanide that is increased greatly: from the product ofthe dissociation constant and the velocity constant for the formation of catalase- cyanide a value of 44 sec.-' is computed for the velocity constant for the dissociation of bacterial catalase-cyanide. This is about eleven times that for erythrocyte catalase-cyanide and is comparable to the dissociation velocity of oxyhaemoglobin. With higher concentrations of hydrocyanic acid (approx. 20 AtM), the combination ofcyanide with the free haematin groups of the bacterial catalase- hydrogen peroxide complex is clearly shown in Fig. 8A on a slow-time scale and in Fig. 8B on a fast- time scale. These measurements were made in the rapid-flow apparatus. On initiation of the flow, the catalase-cyanide remaining from the previous ex- periment is flushed out ofthe observation tube and is replaced by fresh catalase, hydrogen peroxide, and hydrocyanic acid as indicated by the abrupt de- crease in optical density. The catalase-hydrogen peroxide complex is not measured at this wavelength because it forms very rapidly, and because 425 mit. is near the isobestic point for catalase and catalase- hydrogen peroxide (Fig. 3). Thus the extinction in the observation tube is nearly the same as that offree catalase. The rapid upward deflexion (D2) as the flow stops is caused by the combination of catalase and cyanide, and this reaction, according to Fig. 8B, is complete in about 0 1 sec. But Fig. 8A clearly shows that the upward deflexion continues at a slower rate until a constant value El is reached. The slow re- action is caused by the decomposition of catalase- hydrogenperoxide which liberates catalase haematin 200 Initial [hydrocyanide acid] QfM) Fig. 7. Titration of bacterial catalase with hydrocyanic acid. The values of the dissociation constant K, are com- puted for the various points. Final concentrations: 0 4,uM-bacterial catalase, 0 0lm-phosphate, pH 7-0; A=435 mp. to combine with cyanide. Thus E2/E, is a measure of the catalase haematins not bound to hydrogen per- oxide in this complex, and 1- E2/E, ameasure of the catalase haematins bound to hydrogen peroxide. Fig. 8 is, therefore, clearly in agreement with the previous tests which show that not all the catalase haematins are bound to hydrogen peroxide in this complex. Fig. 9 represents the effect of cyanide concentra- tion upon the value of 1- E2/E, and clearly shows a decrease in this quantity with increasing cyanide concentration. This demonstrates that cyanide may compete with peroxide for catalase haematin. How- ever, this competition interferes with the accurate measurement of the maximum value of 1- E2/Ej, which would be obtained at very low cyanide con- centrations. Such data are difficult to obtain with bacterial catalase because of the large dissociation constant of the cyanide complex (22 x 10-6M) which necessitates a relatively large excess ofhydro- VoI. 46 407 B. CHANCE AND D. HERBERT cyanic acid to give readily measurable amounts of catalase cyanide. Therefore the maximum value of 1- E2/E1 is obtained by extrapolation of the data of Fig. 9 to zero hydrocyanic acid concentration, as indicated by the dotted portion ofthe curve. A value ofabout 0-39 is obtained, which corresponds to about 1-6 haematins bound to hydrogen peroxide for a four-haematin catalase. Flow velocity trace sec. Bacterial catalase. _ cyanide compound Bacterial catalase \ E hydrogen peroxide \ compound v S Bacterial catalase T cyanide and E2 hydrogen2 peroxide L compounds Time after flow stops (sec.) 0 1 (A) (B) Fig. 8. The simultaneous reactions of hydrogen peroxide and hydrocyanic acid with bacterial catalase on a slow- time scale (A) and on a fast-time scale (B). Final con- centrations: 0-045,uM-bacterial catalase, 200 ps-hydrogen peroxide, 20 /.M-hydrocyanic acid, 4 mM-methanol, 0-01M- phosphate, pH 7-0; A=435 mp. Iz LU 4.0 20-~~ 0 25 so Initial [HCN] (pLM) Fig. 9. The relation between the quantity 1 - E2/EL (see Fig. 8) and the initial hydrocyanic acid concentration. The decrease of 1 - E2JEL with increasing hydrocyanic acid concentration illustrates competition between per- oxide and cyanide for catalase haematin. Conditions as in Fig. 7. The fact that it is necessary to obtain the value of 1 E2/E1 by extrapolation in the case of bacterial catalase shows that the definition of the experi- mental conditions for a successful experiment for erythrocyte catalase by this method (Chance, 1949c) is inadequate for bacterial catalase. We can point to one inadequacy ofthe previous analysis: the criterion of whether cyanide would displace hydrogen per- oxide from complex I was based upon the initial hydrogen peroxide concentration. However, the initial hydrogen peroxide concentration will have been greatly reduced by the catalatic reaction by the timethe cyanide begins to combinewith the catalase, and competition may indeed occur with more dilute cyanide than would otherwise be expected. This situation is aggravated by the fact that bacterial catalase has a higher activity towards hydrogen per- oxide, and a larger dissociation constant of the cyanide complex, than has erythrocyte catalase. It is therefore recommended that the relation between 1-E2/E1 and cyanide concentration be investigated for each new catalase for which this test is used. The relation between the kinetics of disappearance of hydrogen peroxide and the kinetics of the catalase- hydrogen peroxide complex Numerous attempts have previously been made to devise methods for the measurement of the kinetics of the catalase-hydrogen peroxide complex at the same time as the kinetics of the disappearance of hydrogen peroxide (Chance, 1947). By the develop- ment of suitable electronic circuits and a stopped- T me (sec.) -_ _n m 1 Flow velocity " J ' ** WSUlrA 1rE\ cacatawae (1-202] 0 ~ aalase-o J t/H~<-2o2 com plex Wavelength (mp.) 230 405 Fig. 10. Illustrating the relationship between the kinetics of disappearance of hydrogen peroxide measured at 230 mI. and the kinetics of the bacterial catalase- hydrogen peroxide complex measured at 405 m,. Modified Beckman spectrophotometer, stopped-flow method, cell depth 1 cm. Time markers are at 1 sec. intervals. Final concentrations: 0-07 /LM-bacterial cata- lase, 80uMm-hydrogen peroxide, 8 mm-methanol, O-O1M- phosphate, pH 7-0. (Exp. 416b.) flow method suitable for measurements in the ultra- violet region, the rapid disappearance of hydrogen peroxide can be measured with a fairly rapid re- sponse. This stopped-flow apparatus has an observa- tion chamber with an optical path of 1-0 cm. and permits the measurement of the kinetics of the catalase-hydrogen peroxide complex in very dilute solutions (007 pM). A preliminary record of this type is shown in Fig. 10 and represents the performance obtainable with the present technique operating near the limit ofitssensitivity. At 230 m,u. isregisteredthe kinetics of the disappearance of hydrogen peroxide, and the description given for Fig. 2 applies here except that the time scale is more rapid. At 405 mI,., the abrupt 40nw8 0-0714zM_ <_ +-1--% A .; 0 on..- -, COMPOUNDS OF BACTERIAL CATALASE AND PEROXIDES for formate, methanol and ethanol are 150, 54 and 11 M-1 sec.-' respectively. The agreement with the data of Fig. 13 is fair except in the case ofmethanol where the value is smaller for the alkyl hydrogen peroxide complex. bo 0 i I I I Log (acceptor] Fig. 14. The second-order reaction of bacterial catalase- hydrogen peroxide complex I with various acceptors. The 450 straight lines through the experimental points demonstrate the second-order reaction mechanism. Acceptor concentrations are given in terms of molarity and the units of the velocity constants are m-1 sec.-'. Final concentrations: 0-185 ,uM-bacterial catalase, 80 uM- hydrogen peroxide, 0-01 M-phosphate, pH 7-0; A =405 mA. The reaction of the primary catalase-alkyl hydro- gen peroxide complex with formaldehyde (probably as methylene glycol) proceeds satisfactorily and k4= 270M-1 sec.-'. The difficulties in the measure- ment of the reaction of erythrocyte catalase- hydrogen peroxide with formaldehyde and erythro- cyte catalase-methyl hydrogen peroxide with nitrite are recited elsewhere (Chance, 1950b) and are veri- fied in these experiments. DISCUSSION The great similarity of erythrocyte and bacterial catalase indicated by the work of Herbert & Pinsent (1948) is apparently restricted to those properties of the enzymes which are principally determined by the chemical nature oftheir prosthetic groups and by the size of their protein components: haematin content, light absorption, sedimentation constant, etc. In addition, their enzymic functions and specificities are found to be generally the same. Generally speaking, these studies extend the simi- larity of the two enzymes to the properties of their enzyme-substrate compounds. Bacterial catalase forms with hydrogen peroxide and with methyl or ethyl hydrogen peroxide primary and secondary compounds which have absorption spectra that are similar to those ofthe primary erythrocyte catalase- peroxide compounds. In addition, these compounds form rapidly and oxidize alcohols and related com- pounds. It is of special interest to note that the oxidation of acceptors by bacterial catalase and per- oxides apparently involves only the green primary complex, in accord with previous studies of mam- malian catalases. Superficially, the enzymes might be said to be practically identical. But Herbert & Pinsent (1948) found the value of activity (Kat. f.) towards hydrogen peroxide is larger for bacterial catalasethan forerythrocyte catalase. This difference has been studied in some detail and is verified by these data which show that the velocity constant (kg) for the disappearance of hydrogen peroxide in the presence of bacterial catalase is 5-3 x 107 M-1 sec.-' compared with 3-5 x 107 for erythrocyte catalase. This difference is related to two other differences revealed by these experiments: (1) The number of hydrogen peroxide molecules per bacterial catalase molecule in the primary complex is 1-6 + 0-1, com- paredwith 1-0 ± 0-1 foundforerythrocytecatalaseby similar methods. (2) The reaction-velocity constant (kl) for the combination of bacterial catalase and hydrogen peroxide is about 6 x 107M-1 sec.-' com- pared with about 3 x 107 for erythrocyte catalase under similar conditions. Thus the greater activity of bacterial catalase maybeattributedtothemorerapid combination with hydrogen peroxide which in turn causes a higher steady-state concentration of the catalase-hydrogen peroxide complex. Since the rate. of disappearance of hydrogen peroxide is propor- tional to the steady-state concentration of the inter- mediate complex, the activity towards hydrogen peroxide, as measured in terms of Kat. f. or k1, would be expected to be larger than the value for erythrocyte catalasewhichhas a smaller steady-state concentration of the intermediate complex. The relation between these factors is expressed quantitatively as follows. The mechanism for cata- lase action is generally represented: (2) k, E + S = ES, (e -p) (x) (p) k4 ES+S -- E+Q. (p) (x) The lower-case letters under the symbols of equations (2) 4nd (3) represent their concentrations at any time t. The kinetic equations are: dp = k,x(e -p) -k4xp, (4) dx _ =-k,x(e-p)-k4xp. (5) In the steady state dp/dt= 0, and equation (4) is solved for ple: p 1 e 1 + k4/k, (6) 27-2 (3) 411VOI. 46 B. CHANCE AND D. HERBERT This quantity (ple) is the fraction of the enzyme bound as enzyme-substrate complex, and is 1-0/4-0 for erythrocyte catalase and 1-6/4.0 for bacterial catalase. Equation (6) shows that the quantityp/e is independent ofx but dependent upon k4/kl. By sub- stitution of these values of p/e, equation (6) also shows that the ratio of the values of k4/k1 for ery- throcyte and bacterial catalases must be 3-0:1-5. Assuming that the values of k4 are the same for bacterial and erythrocyte catalases, then the ratio of the values of k, is 2: 1, in agreement with the experimentally determined ratio (6 x 107: 3 x 107). The effect ofple upon the activity of the two cata- lases as measured in terms of the rate of disappear- ance of hydrogen peroxide (dx/dt) is shown by adding equations (4) and (5) dp dx -+d=-2k4xp. (7) For the steady state dp/dt= 0 and dx d =-2k4xp. (8) The velocity of disappearance of hydrogen per- oxide is directly proportional to p, the molar amount of catalase combined as enzyme-substrate complex. Since in the previous discussion it is assumed that k4 is constant, and the value of p is found experi- mentally to be 1-6 times as great for bacterial cata- lase as for erythrocyte catalase, the relative values of their activities (dx/dt), computed as Kat. f. or k1 (equation 1) would be expected to differ by a factor of 1*6, as indeed they very nearly do (53_ 10_= 15'L\3.5 x 107 Thus the consecutive reaction mechanism affords a satisfactory basis for the observed differences of the erythrocyte and bacterial catalase kinetics. How- ever, we are not yet able to obtain satisfactory agreement between the absolute values of p/e, kl, k4 and k1 on the basis of the simple mechanism. It is of considerable importance in the theoretical analysis of catalase action to discover a catalase with significantly more than one hydrogen peroxide molecule bound per catalase molecule. Bacterial catalase, with 1*6 hydrogen peroxide molecules so bound, provides direct evidence against a particular theory ofcatalase actionandsubstantially eliminatbs it from consideration. This theory postulates that catalases, on combination with one molecule of hydrogen peroxide, acquire 'special properties' which prevent the combination of further molecules of hydrogen peroxide (Chance, 1949e). This theory actually fits most ofthe data on erythrocyte catalase, but may now be rejected since with bacterial cata- lase 1*6 molecules of hydrogen peroxide are bound. The altemative theory that the binding of hydrogen peroxide molecules is purely a statistical effect in the consecutive reactions ofhydrogen peroxide with free catalase and catalase-hydrogen peroxide is therefore retained as the most promising explanation for cata- lase action. Whereas bacterial catalase is relatively more active than erythrocyte catalase towards hydrogen peroxide, it is relatively less active with larger sub- strate and acceptor molecules. The decrease in the reactivity of erythrocyte catalase towards sub- strates and acceptors of increasing size has already been commented upon (Chance, 1949d). However, the effect is much more striking with bacterial cata- lase. The velocity constants for its reaction with methyl and ethyl hydrogen peroxide are each about 10 times smaller than the values for erythrocyte catalase. And in the reactions with acceptors, nitrite reacts more rapidly, while propanol, which reacts at an appreciable rate with erythrocyte catalase-peroxides, gives a scarcely measurable rate Table 2. A compariaon of the velocity constant8 for the reactions of bacterial and erythrocyte catala8e8 with 8ub8trates and acceptor8 (Velocity constants for the combination of enzyme and substrate. Units M-1 sec.-l) Substrate Erythrocyte catalase Bacterial catalase Hydrogen ... peroxide 3 x 107 6 x 107 Methyl hydrogen peroxide 8 x 105 2-2 x 105 Ethyl hydrogen peroxide 20 x 103 2-8 x 103 (Velocity constants for the reaction of the primary enzyme-substrate complexes with acceptors. Units M-1 sec.-') Acceptor ... ... Nitrite* Formaldehyde Methanol Ethanol Formate* Propanol Erythrocyte catalase-hydrogen 2000 1000 1000 470 17 peroxide, I Bacterial catalase-hydrogen peroxide, I Erythrocyte catalase-methyl hydrogen peroxide, I Bacterial catalase-methyl hydrogen peroxide, I 2800 91 1400 . 910 270 54 13 175 0 025 910 460 11 150 * pH =7-0. I950412 COMPOUNDS OF BACTERIAL CATALASE AND PEROXIDES with bacterial catalase-peroxides. These various velocity constants are summarized in Table 2 and amplify the comparison considerably. Reasoning along the same lines as with erythrocyte catalase, it is concluded that the haematins of bacterial catalase are even less accessible than those of erythrocyte catalase, and only very small molecules are suitable as acceptors. Following this idea further, it is pro- posed that the greater proximity ofprotein and pros- thetic group of bacterial catalase causes interactions which result in a higher activity towards hydrogen peroxide. In other words, the proximity of protein and prosthetic group in bacterial catalase enhances its activity towards small molecules but results in steric effects which abruptly decrease the activity towards larger molecules. Thus rather indirect evidence is presented for the spatial interaction of protein and prosthetic group resulting in increased activity and a higher specificity of the enzyme. Further studies ofrelated haemoproteins are required to extend this theory. SUMMARY 1. Bacterial catalase forms primary and second- ary enzyme-substrate complexes with hydrogen per- oxide, methyl hydrogen peroxide and ethyl hydro- gen peroxide. 2. The spectra of the bacterial and erythrocyte catalase-methyl hydrogen peroxide complexes in the region of the Soret band are nearly identical. How- ever, a quantitative difference exists between the Soret bands of the primary hydrogen peroxide com- plex of bacterial and erythrocyte catalases (see below). 3. The velocity constant* for the decomposition of hydrogen peroxide in the presence of bacterial cata- lase is 5-3 x 107M-1 sec.-' for a four-haematin cata- lase, andmay be compared with the value of 3-5 x 107 for erythrocyte catalases; i.e. the ratio ofthe velocity constants (or of the Kat. f. values) for the two en- zymes is 1-5:1. 4. The bacterial catalase-hydrogen peroxide complex consists of 1-6 + 0-1 hydrogen peroxide molecules per bacterial catalase molecule, as de- termined by spectrophotometric measurements of catalase-methyl hydrogen peroxide and cyanide compounds. This value may be compared with that of 1 -0 + 0-1 found for erythrocyte catalase on the basis of methyl hydrogen peroxide data. 5. The velocity constant for the combination of bacterial catalase with hydrogen peroxide is about * Velocity constants are measured at 20-25'. 6 x 107M-1 sec.-' and the reaction is shown to be of thesecond orderup to 20 ,uM-hydrogen peroxide. This value may be compared with that of about 3 x 107 for erythrocyte catalase. 6. On the basis of the mechanism for catalase action which involves consecutive reactions of hydrogen peroxide with catalase and with the cata- lase-hydrogen peroxide complex, the greater activity of bacterial catalase compared with erythrocyte catalase may be quantitatively attributed to the more rapid combination of bacterial catalase with hydrogen peroxide to form the bacterial catalase- hydrogen peroxide complex. The consequence ofthis more rapid reaction is a larger steady-state concen- tration of the bacterial catalase-hydrogen peroxide complex and a higher velocity constant for the dis- appearance of hydrogen peroxide. 7. The velocity constants for the combination of bacterial catalase with methyl or ethyl hydrogen peroxide are 2-2 x 105 and 2-8 x 103M-1 sec.-' respec- tively. These values are about 10 times 8lower than the corresponding values for erythrocyte catalases. 8. The primary bacterial catalase-peroxide com- plexes react with acceptors according to the mech- anisms found to obtain for the corresponding ery- throcyte catalase-peroxide complexes; only the green primary complexes appear to be involved in these reactions. The specificity appears to follow an identical pattern; velocity constants are evaluated for nitrite, formaldehyde, formate, methanol, ethanol and propanol. 9. Bacterial catalase and hydrogen peroxide oxidizes nitrite more rapidly than does erythrocyte catalase. Also bacterial catalase is relatively more active towards formate and formaldehyde with respect to methanol and ethanol than is erythrocyte catalase. 10. In the reactions of bacterial catalase with sub- strates or acceptors, the values of the reaction velocity constants are dependent on the size of the substrate or acceptor molecule to a much greater extent than was found to be the case with erythro- cyte catalase; large molecules react much more slowly than small ones. This greater dependence is interpreted to indicate that the haematins of bac- terial catalase are 'buried' in the protein molecule to a greater extent than are the haematins of ery- throcyte catalase. Nevertheless, the haematins of bacterial catalase react with hydrogen peroxide more rapidly than do the haematins of erythrocyte cata- lase. It is suggested that the proximity ofthe protein groups to the haematins of bacterial catalase en- hances their activity towards molecules to which the haematins are accessible. VoI. 46 413