A Kinetic Study of the Reaction of Horseradish Peroxidase with Hydrogen Peroxide1 B. B O L M A N G., ~A. NEWBLL, AND M. D. THURLOW Can. J. Biochem. Downloaded from www.nrcresearchpress.com by University of Winnipeg on 11/02/18 For personal use only.
Departme~ztof Clremistry, Utriversity of Lethbridge, Lethbridge, Alberta T I K 3M4 AND
H. B. DUNFORD Departmertt oJ C/re~nistry,Utmiversity of Alberta, Ed~noirtotr,Alberta T6G 2 E l Received August 13, 1974 Dolman, D., Newell, G. A., Thurlow, M. D. & Dunford, H. B. (1975) A Kinetic Study of the Reaction of Horseradish Peroxidase with Hydrogen Peroxide. Carm. J. BiocEterrz. 53,495-501 A kinetic study has been carried out over the pH range of 2.63-9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound 1 of the enzyme. Analysis of the results. indicates that there are two kinetic influencing, ionizable groups on the enzyme with pK, values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H 2 0 z . A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W. D. & Dunford, H. B.: Biochemistry 7, 2054-2062 (1968)) has been extended down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pK, values of 2.9 and 3.9. Dolman, B., Newell, G. A., Thurlow, M. D. & Dunford, H. B. (1975) A Kinetic Study of the Reaction of Horseradish Peroxidase with Hydrogen Peroxide. Cmm. J . Biochetn. 53,495-501 Nous avons poursuivi l'ktude cinitique, h des pH variant de 2.63 B 9.37, de la rdaction de la peroxydase de raifort avec le geroxyde d'hydrogkne pour former le composd 1 de l'enzyme. L'analyse des rCsultats montre que I'enzyme comporte deux groupes ionisables qui influencent la cindtique et dont Ies valeurs de pK, sont de 3.2 et 3.9. La protonation de ces groupes diminue la vitesse de rdaction de l'enzyme avec le H z 0 2 . Pour compldter un travail antdrieur (Ellis, W. B. & Dunford, H. B.: Biochenaistry 7, 20542062 (1968)) nous avons CtudiC, B pH 2.55, la cinCtique de la liaison entre le cyanure et la peroxydase du raifort. E'analyse des rdsultats montre aussi que l'enzyme contient deux groupes ionisables, importants du point de vue cinCtique et dont les valeurs de pK, sont de 2.9 et 3.9. [Traduit par Be journal]
Introduction The general scheme by which horseradish peroxidase (HRP) catalyzes the oxidation by hydrogen peroxide of a wide variety of' organic compounds was established by the work of Chance (1) and George (2, 3) and is presented in E ~ s 1-4. .
[I] [2] [3]
HRP
+ H202
--+
HRB-I
+ AH2 --+ HRP-I1 + AHe HRP-I1 + AH2 HRP + AH'
HRP-I
--+
I31
2AH' --+ products Here HRP refers to native enzyme, HRP-I and HRP-I1 are oxidized forms of the enzyme called -
ISupported financially by the University of Lethbridge Research Committee. 2To whom correspondence should be addressed.
compounds I and I1 and containing, respectively9 two and one oxidizing equivalent relative to the native enzyme, and AH2 is an oxidizable substrate. Recently, considerable information has become available about the fate of the hydrogen peroxide (4-6) and about the accompanying changes likely to occur in the hemin prosthetic group (7) during the catalytic cycle of peroxidases. In addition, as a result of a number of studies of the pH dependence of the kinetics of reaction 2 (8-1 1) and of reaction 3 48-10, 12-23) using various reducing substrates, infornlation has been obtained on the involvement of ionizable groups on the enzyme in reactions 2 and 3. Less information is available about the involveIllent of ionizable groups in the formation of compound I from native enzyme (reaction I). Studies of the pH dependence of the kinetics of
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396
C.4N. J. BLOCHELM. VOL. 53, 1975
binding of fluoride (15, 16) and cyanide (16) to HRP have indicated the presence sf three kinetically important ionizable groups on the enzyme. This paper is concerned with an extension sf the kinetic studies to reaction 1 itself, which we have studied over the pH range of 2.63-9.37. Also, the kinetics of the binding of cyanide to HRP has been studied down to pH 2.55 to extend the results of the previous study (16) which \vent only as far as pH 4.2.
Materials and Methods Horseradish peroxidase was purchased from Boehringer-Mannheim Corp.. New York (PN3 approximately 0.6), aild was further purified on a column of Sephadex C-50 cation exchange gel as described previously (8). Fractions with PN greater than 2.8 were kept for kinetic experiments and were stored at 4 "C as an ammonium sulfate precipitate. The precipitate was dissolved, and dialyzed against distilled water prior to use. The concentration of HRP was determined spctrophotometrically using a molar adsorptivity of 9.1 X 104h f - I can-' at 403 mm (171, Hydrogen peroxide, potassium cyanide, and all buffer materials were either of reagent or AR grade and were used without further purification. Doubly distilled water was used for all solutions. Hydrogen peroxide solutions were prepared by successive dilutions of a 30':; solution. On the day of an experiment, a lo-* M so1ution of H 2 0 2was prepared and analyzed according to the method of Ovenston and Rees (18) at the beginning and at the end of a set of kinetic experiments. Decomposition of the H20? during the experiments (a period lasting from 1 to 3 h) generally was less than 17;. The reaction solutions containing HzOz were made up by appropriate dilutions of the lo-* M s o l u t i o ~immediately ~ prior to a kinetic run and the concentrations calculated rasing the dilution factor. The reactions were followed spectrophotometrically using a Dureum-Gibson stopped Mow spectrophotometer thermostated at 25 i 0.5 'C.All kinetic runs were performed in solutions of final total ionic strength 0.1 1 M with the buffer contributing 8.01 A4 and potassium nitrate contributing 0.10 M. The enzyme solutions used in the stopped flow experiments contained potassium nitrate but no buffer, whereas the hydrogen peroxide and potassium cyanide solutions contained both potassium nitrate and bufffer. Buffer was not added to the enzyme solutions, iaa order to avoid denaturation at low pH of the enzyme prior t.o mixing of the reaction solutions. This allows measurements to be made down to about pH 2.5 where the rate of denatliratisn approaches the rate of reaction. This technique assumes that the half-life for proton equilibration is much shorter than the half-life of the reaction being studied. In the hydrogen peroxide study, the reaction mixtures contained approximately 3.5 X lW7 A4 HRP and conT N , purity umber defined as the ratio sf absorbances at 403 and 280 nm.
centrations of H2Q2varying from 7 X 10-7 !W (pH 4-10) to A4 (pH 3.4). At this concentration of HRP, the absorbaiace change during the reaction was less than 0.05 and conseyue~ltlythe approximation can be made that the absorbance change, L A , is proportionat to the photomultiplier voltage change, AV, without incurring an error of more than 5' ;. These Iow concentrations of HRP were also necessary in the pH range from 4 to 10, in order to keep the reaction slow enough to follow on the stopped flow apparatus. Chance ( I ) has shown that reaction 1 is first order in both HRP and H.02. The reaction traces were analyzed for the second-order rate constant, k,,,,,, at a particular pH value, using the following integrated second-order rate law.
in this and subsequent equaThe subscripts o, t, and tions refer to initial, intermediate, and complete stages of reaction. The initial concentration of H 2 0 2and HRP were known and the terms [N202],and [HRP], were calculated from the oscilloscope traces of photomultiplier voltage 4483 nm) using the following equations:
where V refers to the photomultiplier voItage on an arbitrary scale. To obtain V oit was necessary to make a small extrapolation to account for the amount of reaction that occurs during the dead-time of the apparatus (3 ms). The product of reaction 1 , compound I , was stable enough on the time scale of the reaction that V , could be obtained by triggering the oscilloscope trace 1-2 s after the initial trace. It was assumed that the reaction is irreversible and that a 1 :1 stoichiometry exists between MRP and H20y. In the study of the kinetics of cyanide binding to HRP. the concentratiotas of MNOa and buffer were the same as in the hydrogen peroxide study. The HRP concentration was approximately 6 X 10-7 M and the reaction was followed at 422 nm with an absorbance change during reaction of approximately 0.07. The concentration of KCN varied from about 2 X 1 W4 i%f (pH greater than 4) to 1 0 - W 6pH 2.55) and was such as to drive the reaction to completion (86). Below pH 4.2, the association equilibrium constants for cyanide with HRP are not kimown and cannot be measured readily, due to competing denattiration of the enzyme. In this pH region, the cyanide concentration was increased to such a level as to keep the half-life of the reaction in the range from 50 to 80 ms. The reaction traces showed the same overall voltage change over the entire pH range of the study, indicating that errors due to possible lack of complete complexing at low pH were minimal. In all cases, the concentration of cyanide was at least 100-fold that of the enzyme, and consequently the reactions followed first-order kinetics. The reaction traces were analyzed according to the first-order integrated rate equation with photomultiplier voltage changes converted
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DOLMAN ET AL.: HORSERADISH PEROXIBASE WITH HYDROGEN PEROXIDE
BUFFERS 0
cescodyBic acid -sodium cacodylate
KH2P04 - Na2HP04 A
boric acid
- sodium borate
s citric acid- sodium citsate
3
5
7
PH
Fa@. 1. Semilogarithmic plot of k,,, vs. pH for the reaction of WRP with H202.The curve is that one predicted by Eq. 9 using the foiliowing values: k l , 1.78 >< 10: M-I s-l; Ki, 5.64 X 10-r; K?, 1.29 X los4. Each point represents the average of three determinations of k,,, made using the same reactant solutions.
to absorbance changes before analysis of the data. The second-order association rate constant, k,,,, at a particuIar pH was calculated from the observed first-order rate constant, k,,,, and the total cyanide concentration, [CN],. using Eq. 8.
PI
k,,,
=
k,,,/ K N I T
Below pH 4, the cyanide concentrations used were large enougll to overcome the buffering capacity of the solutions, and consequently the cyanide was neutralized by adding an amount of nitric acid equivalent to the amount of KCN in solution. Bn addition to the rapid first-order reaction, corresponding to cyanide binding to HRP and with half-life ranging from 50 to 130 ms depending on reaction conditions, there was also observed a mucla slower reaction of half-life about 4 s at pH values greater than 4. This slow reaction exhibits a much smaller absorbance change than the more rapid reaction, and was not observed in the original study (16) carried out using lower cyanide concentrations. It was not studied further. The pH measurements were made with an Orion model 801 digital pH meter with a Fisher universal glass electrode and calomel electrode. Fisher certified buffer solutions were used for calibrating the electrodes. The pH values of the reaction mixtures were not measured directly but by measuring the pH of solutions made from the same K N 0 3 and bufler stock solutions, and with the same dilutions as the reaction mixtures but lacking in reactants.
The accuracy of the rate constants, k,,,,,, obtained for the reaction s f hydrogen peroxide with horseradish peroxidase is limited due to a
number of factors. including the uncertaintv in the molar of HkP(91, of dilute hydrogen peroxide solutions, and the approximation regarding absorbance and photomultiplier voltage mentioned above. As a result, the rate constants are probably uncertain to within 25% or possibly more. his large uncertainty, however, presents no real problem since kinetically important ionizable groups generally produce large changes in rate constants. The second-order rate constants, k,,,,, for reaction of HRP with H2Q2 obtained over the pH range of 2.63-9.37 are plotted sernilogarithmically in Fig. 1. Chance and coworkers (19) have previously measured the rate constant for the reaction of HRP with H 2 0 2and obtained a value of 1.15 X B07M-hs-' at an unspecified but presumably neutral pH. This compares with a value around 1.8 X 107M-I s-I at neutral pH obtained in this work. At the outset of this work, it had been hoped to make rate measurements up to a pH of at least 11, a region in which large changes occur in the spectral (17, 20), kinetic (16), and magnetic (21) properties of the enzyme. These changes have been assumed to result from ionization s f a water molecule exhibiting a p& of 18.8 (16) occupying one of the two axial coordination sites of the ferric ion. It was found, however, that when sate
498
CAN. 9.
measurements were attem~ted above DH 9.5 that the reaction traces became complex and could not be fitted by a second-order rate equation. The reaction traces exhibited an initial rapid voltage change, the amplitude of which increased with increasing pH, followed by a slower voltage change. Investigation of the reaction of the free enzyme with bufler or hydroxide solutions of pH greater than 10, in the absence of hydrogen peroxide, showed that the initial rapid reaction was caused by the pH change alone and was not due to reaction with hydrogen peroxide. As a result of this complication, no measurements of the rate of reaction 1 were made above pH 9.5. It is apparent from the plot of k,,,, vs. pH in Fig. B that ionizations are occurring in the pH range from 2.5 to 4.0 that affect the rate of reaction. Furthermore, since the slope of a plot of log k,,,, vs. pH is greater than H at low pH, there must be at least two ionizable groups involved. The simplest scheme which will account for the data plotted in Fig. B is shown below in Scheme 1.
VOL. 53, 1975
The data were analyzed for the best values of kl, K I , and K2 by the method of least mean squares using Eq. 9. In the analysis, the deviations were weighted by the factor 1 /k,,,,, in order to give equal weight to each data point. The values obtained are: for kr, 1.78 X 107M-I s-I; for KI, 5.64 X 10-2 and for K2, 1.29 X The line drawn through the data in Fig. B is obtained from Eq. 9 using these values of the parameters. A scheme kinetically equivalent to Scheme 1 involving reaction of EH with H02- can be eliminated because it leads to a rate constant for this reaction far in excess of the diflusion controlled 1imit . The results of the study of the binding of cyanide to horseradish peroxidase are plotted semilogarithmically in Fig. 2. Also indicated in Fig. 2 are the results of the previous study by Ellis and Dunford (16). There is a discrepancy between the two sets of results which cannot be accounted for. Tt should be noted in this connection that the two studies were carried out under different conditions. In this study, large concentrations of cyanide were used to drive the reaction to completion, whereas in the previous study, lower concentrations of cyanide were used and produced an equilibrium mixture containing appreciable amounts of uncomplexed as well as complexed enzyme. The previous work was also carried out on an enzyme preparation that was less pure (PN 0.9) than the present. The previous study (16) showed that in the reaction of cyanide with HWP there are three In Scheme 1, protons have been left out for kinetically important ionizations on the enzyme simplicity, E, EH1, and EKE2 refer to successively with pK, values of 10.8, 6.4, and 4.1. The protonated species of the enzyme, the ionization ionization with pK, of 6.4 has very little effect on equilibria of which are related by the ionization the rate of the reaction. The present study was constants Kg and KZ;kl is the pH independent confined to the pH region 2.55-5.5 where the rate constant for the reaction of E with H,O,. effects of the ionizations with pK, values of 6.4 The rate constants for the reactions of HzOZ and 10.8 make no important contribution to the with EHI and EH2 are assumed to be effectively observed kinetics and, as a consequence, analysis zero, and equilibration among the protonated of the results obtained in this work does not take species of enzyme is assumed to be maintained into account these ionizations. As with the results of the reaction of hydrogen throughout the reaction. This scheme leads to peroxide with MWP, the results of the reaction sf Eq. 9 for kaPp4. cvanide with WRP indicate at least two isnimble k~ boups influencing the kinetics below pH 4. [g] kapp = Reaction Scheme 2, which is directly analogous (1 4- [H+]/Kz [H+]"KIKz) to Scheme 1, can be used to account for the 4Activity coefficients have been neglected in deriving observed ka,, values for cyanide binding. %heme 2 leads t o Eq. 9 for k,,,, and this equation was Eq. 9 and in calculating [H+] from measured p~ values. I
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BHOCHEM.
+
1
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DOLMAN EY AL.: HORSERADISH PEROXIBASE W I T H HYDROGEN PEROXIDE
this study
Ellis and Dunford (16)
FIG. 2. Semilogarithmic plot of k,,, vs. pH for the reaction of WRP with WCN.The curve is that one predicted by The points for this study Eq. 9 using the following values: k l , 1.19 X lo6M-I s-l; K 1 , 1.24 X Kz, 1.10 >< are the average of two determinations made using the same reactant solutions, carried out in citric acid - sodium eitrate buffers except for the point at pH 5.50 where caeodylic acid - sodium cacodylate was used.
used to analyze for the values of kl, K1, and K2 trolled rate constant expected for a reaction in the same manner as before. between a protein and a small n~olecule(22) and must be regarded as an unlikely alternative. In this regard, Erman, in studying the binding of fluoride (23) and cyanide (24) to cytochrome c peroxidase (CcP), has argued that the neutral forms of these weakly basic ligands bind to the heme iron in peroxidases. In fact, the rate constants for the binding of HCN to the unprotonated forms of the two enzymes are the same, 1.2 X 105M-I s-I for HRB and I. 1 X 105 M-I s-I for CcP (24). The results of the analyses of the two studies The best values obtained from the analysis of are collected in Table I. Of particular interest are the data are: for kl, 1.19 X 105 M-I s-I; for K1, the values of K1 and K2for the ionizations on the 1.24 X 10-3; and for K2, 1.10 X The line free enzyme. The two K2 values are identical drawn through the data in Fig. 2 is obtained from within reasonable experimental error. The two Eq. 9 using these values of the parameters. K1 values differ by a factor of about two, and it is Scheme 2 is an extension of the original kinetic difficult to decide whether or not this difference scheme of Ellis and Dunford (16) in which the is significant. The precision of the two data sets, ionization of EH1 (pK, 3.9) in this study corre- as indicated by the standard deviations, is such sponds to the ionization of the group with pK, that the two K1 values are different; however, 4.1 in their study. As was the case in the original more realistic error limits are undoubtedly much study, there is a kinetically indistinguishable larger than the standard deviations. It is quite alternative mechanism involving reaction of CN- possible that there are systematic errors present with EHL.Based on a pK, value of 9.0 (16) for in either or both sets of kinetic data that could HCN at an ionic strength of 0.1 I, the rate con- account for the observed difference in K1 values. stant for this reaction is 1.3 X 1010M-' s-I. This This is particularly likely at low pH where r diffusion eon- denaturation of the enzyme is rapid. The possivalue is at the upper limit f ~ the
CAW. J. BIOCHEM, V O t . 53, 1975
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TABLE 8 . Values for the parameters of Eq. 9 obtained from analysis of the data in Figs. 1 and 2" Reaction
k l , M-l s-I
KI
HRP f Hz82 (Schetnel)
(1.78+0.$3)X107
(5.64+0.42)Xl0-"(%.29+0.08)XleB-'
HRP f HCN (Scheme 2)
(1 .19+0.02)X105
(l024kO.07)X10-"1.
Kz
10kQ.$5)X10-2
UThe errors are the standard deviations determined from the least-squares analysis of the data. Actual errors are considerably larger.
bility cannot be dismissed, however, that the two Kl values refer to two distinct ionizable groups. -
Discussion Comparison of the kinetic results over the pH range of 2.5-9.5 for the reactions of H202 and HCN with HRP indicates that the a ~ ~ a r erate nt constants for the two reactions are affected in the same manner by ionizations of specific groups on the enzyme. Both reactions reveal the presence of two kinetically important ionizations on the enzyme with pKLLvalues in the range of 2.9-4.0. The HCN-HRP kinetic results reveal, in addition, a group on the enzyme with a pKa value of 6.4 (16) that is not evident in the H202-HRP reaction, Ionization of this group with pK:, of 6.4 has little eKect on the kinetics of HCN binding to HRP and it was included primarily to satisfy the principle of detailed balancing (16). The similarity of the two reactions in regard to the effect of pH on the kinetics suggests that there are similarities in their mechanisms. A possible explanation for this similarity might be found in the following mechanism (Scheme 3) for reaction of H 2 0 2with HRP.
tional assumption that the concentration of intermediate remains low relative to the concentration of free enzyme, results in Eq. 18 for the rate of compound I formation.
6 101
d[compound I] dt
I P
compound I SCHEME 3
In Scheme 3, E-Fe(H20) indicates the free enzyme with a water molecule coordinated to the ferric ion at one of its axial positions, and E-Fe(H202) indicates hydrogen peroxide coordinated in the same position to the iropi atom. Application of the steady-state assumption to the intermediate E-Fe(Hn02), with the addi-
Thus the two step process leads to a prediction of second-order kinetics, as is observed for compound I fornaation, with k,,, identified with klk2 /(k-1 f k2). The first step in the mechanism outlined in Scheme 3 is analogous to the reaction of HCN with HRP and consequently Bdr in Scheme 3 should show the same pH dependence due to ionizable groups on the enzyme as is found in the HRP-HCN reaction. In turn, the pH dependence of kl, according to Eq. 10, is carried into k,,,,. A more complicated pH dependence of k,,,, than is observed is predicted by mechanism 3, however, if the two ionizable groups corresponding to K I and K2 also affect the rate of the second step, formation of compound I from E-Fe(H203). Support for the above mechanism can be found in the work of Portsmouth and Beal (25) who have found that hydrogen peroxide reacts with deuterohemin to form a product capable sf oxidizing typical peroxidase substrates. The rate of formation sf this product is not first order in the concentration of; H 2 0 2 but rather shows a saturation effect with respect to increasing H2On concentration. This result has been interpreted by Portsmouth and Beal as indicating the formation of a hemin-H202 con~plexas an intermediate en route to formation of the persxidatic product. This, s f course, is a direct analog of mechanism 3.
DOLMAN ET AL.: HORSERADISH PERCBXIDASE WITH HYDROGEN PEROXIDE
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A reasonable proposal for the identity of the pK, values of 3.2 (or 2.9) and 3.9 is that they belong to the carboxyl groups of the porphyrin propisnic acid side chains. Support for such a view is available from the work of Maehly (26) and of Tamura et aE. (27). Maehly showed that protohemin and protoporphyrin both bind to horseradish apopessxidase to produce strong Soret band absorption at 403 nrn, characteristic sf native horseradish peroxidase, whereas protohemin dimethyl ester and protoporphyrin dimethyl ester do not exhibit such adsorption changes. Tarnma el ak. also observed that the apoenzyme has a reduced aEnity for the protohemin dimethyl ester as compared with its affinity for native protohemin. They found that the enzyme reconstituted from apoenzyme and the protohemin dimethyl ester has a much reduced affinity for ligands such as cyanide and a much reduced reactivity towards hydroperoxides. Since protonation of both of the carboxyl groups of the hernin would be expected to produce effects similar to those observed irn the dimethyl ester, it seems reasonable to identify the two ionizable groups found in the MRP-H202 study with the hemin carboxyl groups. The situation is not clear cut, however, since these workers (27) found that protohemin monomethyl ester combines with apoemyrne to form an enzyme reactive towards both cyanide and hydroperoxides. If the kinetically important ionizable groups are indeed the hemin carboxyl groups then this result would lead one to expect the mornoprotonated species, EH1, to be reactive towards hydrogen peroxide, contrary to observation. It is, of course, possible that the species EH1 has substantial b i t reduced reactivity towards H202,as compared with that of E, which was not detected in this study. Although one of the ultimate goals of this type of study is to identify and to determine the role of ionizable groups that participate in the catalytic cycle of the enzyme, this goal is still far from being present no obvious identification of the pKa values 3-2 2-9) and 3-9 found on the free enzyme can be made with pK, values on compounds I and 11 of H W P round in previous siudies (8-14).
50 1
I . Chance, B. (L952) Arch. Biochem. Biopkys. 41, 416424 3* George, P. (1952) A'ature 169, 612-613 3. George, P. (1952) Bioc/zem. J. 54, 267-276 4. Schonbaum, G. R. & Lo, S. (1972) J . B i d . Chem. 247, 3353-3360 5. Hager, L. P., Doubek, D. L., Silverstein, W. M., Lee, T. T., Thomas, .I.A., Margis, J. H. & Martin, J. C . (1972) iia Oxidases c6nd Related Recbos Sj3stems(King, T, E., Mason, H. S. & Iklsrrison: M., eds), pp. 311332, University Park Press, Baltimore, Md. 6. Hager, L. P., Doubek, D. L.; Silverstein, R. M., Hargis. J. I%. & Martin, J. C. (8972) J. Ans. Chem. Soc. 94,4364-4366 7. Dolphin, B., Forman, A., Bsrg, D. C., Fajer, J. & Felton, W. H. (1971) Broc. !Vat/. Acczd. Sci. U.S. 68, 614-618 8. HasinoE, B. B. & Dunford, H. B. ( 1970) Biochemistry 9,4930-4939 9. Cotton, M. L. & Dunford, H. B. (1973) Cliiz. J. Chern. 54, 582-587 10. Roman, W. & Dunford, H. B. (1973) Can. J. Chen~. 51, 588-596 1I . Roman, R.& Dunford, H. B. (1 972) Biochemistry 1' I , 2076-2082 12. Roman, W., Dunford, H. B. & Evett. M. (L971) Can. J . Chern. 49, 3059-3063 13. Critchlow, J. E. & Dunford, H. B. (1972) 9. Biol. Chem. 247, 3703-371 3 14. Critchlsw, J. E. & Dunford, W. B. (1972) J. B i d . Chem. 247, 3714-3725 15. Dunford, H. B. & Alberty, R.A. (1967) Bioelremisfry 6,447-45 1 16. Ellis, W. D. & Dunford, W. B. BL968) Biochenzistly 7, 20562062 17. Keilin, D. & Hartree, E. F. (1951) Bioclaern. J. 49, 88-104 18. Ovenston, T. C. J. & Wees, W. T. (1950) Atza/ysf Lond. 75,204-208 19. Chance, B., Bevault, D.. Legallais, V.. Mela, L. & Yonetani, T. 6 1967) in Fast Rearcfio~msa~zdPrimary Processes i~zCI~emicalKittetics (Claesson, S., ed.), pg. 437-464, Interscience Publishers Inc., New York, W.Y. 20. Ellis, W. D. & Dunford, H. 8.(1969) Arch. Biochern. Biupj1~3.133, 313-317 21. Theorell, W. (1943) Ark. &mi 1Vli/zera6.Geol. 16-4, No. 3 22. Alberty, R. A. & Han~mes,G. G. (1958) J. Phys. Chem. 62, 154-159 23. Erman, J. E. (1974) Biochemistr:,. 13, 34-30 24. Erman, J - E- (1974) Biochemiht~~ 137 39-44 25. Portsmouth. D. & Beal, E. A. (1971) Eur. J. Biochem. 19,479-487 26. Maehly, A. C . (1961) ~ V a t 192,630-632 ~r~ 27. Tamura, M., Asakura, T. & Yonetani, T. (1972) Biochirn. Bioplays. - Acta 268, 292-304 &
Departme~ztof Clremistry, Utriversity of Lethbridge, Lethbridge, Alberta T I K 3M4 AND
H. B. DUNFORD Departmertt oJ C/re~nistry,Utmiversity of Alberta, Ed~noirtotr,Alberta T6G 2 E l Received August 13, 1974 Dolman, D., Newell, G. A., Thurlow, M. D. & Dunford, H. B. (1975) A Kinetic Study of the Reaction of Horseradish Peroxidase with Hydrogen Peroxide. Carm. J. BiocEterrz. 53,495-501 A kinetic study has been carried out over the pH range of 2.63-9.37 for the reaction of horseradish peroxidase with hydrogen peroxide to form compound 1 of the enzyme. Analysis of the results. indicates that there are two kinetic influencing, ionizable groups on the enzyme with pK, values of 3.2 and 3.9. Protonation of these groups results in a decrease in the rate of reaction of the enzyme with H 2 0 z . A previous study of the kinetics of cyanide binding to horseradish peroxidase (Ellis, W. D. & Dunford, H. B.: Biochemistry 7, 2054-2062 (1968)) has been extended down to pH 2.55, and analysis of these results also indicates the presence of two kinetically important ionizable groups on the enzyme with pK, values of 2.9 and 3.9. Dolman, B., Newell, G. A., Thurlow, M. D. & Dunford, H. B. (1975) A Kinetic Study of the Reaction of Horseradish Peroxidase with Hydrogen Peroxide. Cmm. J . Biochetn. 53,495-501 Nous avons poursuivi l'ktude cinitique, h des pH variant de 2.63 B 9.37, de la rdaction de la peroxydase de raifort avec le geroxyde d'hydrogkne pour former le composd 1 de l'enzyme. L'analyse des rCsultats montre que I'enzyme comporte deux groupes ionisables qui influencent la cindtique et dont Ies valeurs de pK, sont de 3.2 et 3.9. La protonation de ces groupes diminue la vitesse de rdaction de l'enzyme avec le H z 0 2 . Pour compldter un travail antdrieur (Ellis, W. B. & Dunford, H. B.: Biochenaistry 7, 20542062 (1968)) nous avons CtudiC, B pH 2.55, la cinCtique de la liaison entre le cyanure et la peroxydase du raifort. E'analyse des rdsultats montre aussi que l'enzyme contient deux groupes ionisables, importants du point de vue cinCtique et dont les valeurs de pK, sont de 2.9 et 3.9. [Traduit par Be journal]
Introduction The general scheme by which horseradish peroxidase (HRP) catalyzes the oxidation by hydrogen peroxide of a wide variety of' organic compounds was established by the work of Chance (1) and George (2, 3) and is presented in E ~ s 1-4. .
[I] [2] [3]
HRP
+ H202
--+
HRB-I
+ AH2 --+ HRP-I1 + AHe HRP-I1 + AH2 HRP + AH'
HRP-I
--+
I31
2AH' --+ products Here HRP refers to native enzyme, HRP-I and HRP-I1 are oxidized forms of the enzyme called -
ISupported financially by the University of Lethbridge Research Committee. 2To whom correspondence should be addressed.
compounds I and I1 and containing, respectively9 two and one oxidizing equivalent relative to the native enzyme, and AH2 is an oxidizable substrate. Recently, considerable information has become available about the fate of the hydrogen peroxide (4-6) and about the accompanying changes likely to occur in the hemin prosthetic group (7) during the catalytic cycle of peroxidases. In addition, as a result of a number of studies of the pH dependence of the kinetics of reaction 2 (8-1 1) and of reaction 3 48-10, 12-23) using various reducing substrates, infornlation has been obtained on the involvement of ionizable groups on the enzyme in reactions 2 and 3. Less information is available about the involveIllent of ionizable groups in the formation of compound I from native enzyme (reaction I). Studies of the pH dependence of the kinetics of
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396
C.4N. J. BLOCHELM. VOL. 53, 1975
binding of fluoride (15, 16) and cyanide (16) to HRP have indicated the presence sf three kinetically important ionizable groups on the enzyme. This paper is concerned with an extension sf the kinetic studies to reaction 1 itself, which we have studied over the pH range of 2.63-9.37. Also, the kinetics of the binding of cyanide to HRP has been studied down to pH 2.55 to extend the results of the previous study (16) which \vent only as far as pH 4.2.
Materials and Methods Horseradish peroxidase was purchased from Boehringer-Mannheim Corp.. New York (PN3 approximately 0.6), aild was further purified on a column of Sephadex C-50 cation exchange gel as described previously (8). Fractions with PN greater than 2.8 were kept for kinetic experiments and were stored at 4 "C as an ammonium sulfate precipitate. The precipitate was dissolved, and dialyzed against distilled water prior to use. The concentration of HRP was determined spctrophotometrically using a molar adsorptivity of 9.1 X 104h f - I can-' at 403 mm (171, Hydrogen peroxide, potassium cyanide, and all buffer materials were either of reagent or AR grade and were used without further purification. Doubly distilled water was used for all solutions. Hydrogen peroxide solutions were prepared by successive dilutions of a 30':; solution. On the day of an experiment, a lo-* M so1ution of H 2 0 2was prepared and analyzed according to the method of Ovenston and Rees (18) at the beginning and at the end of a set of kinetic experiments. Decomposition of the H20? during the experiments (a period lasting from 1 to 3 h) generally was less than 17;. The reaction solutions containing HzOz were made up by appropriate dilutions of the lo-* M s o l u t i o ~immediately ~ prior to a kinetic run and the concentrations calculated rasing the dilution factor. The reactions were followed spectrophotometrically using a Dureum-Gibson stopped Mow spectrophotometer thermostated at 25 i 0.5 'C.All kinetic runs were performed in solutions of final total ionic strength 0.1 1 M with the buffer contributing 8.01 A4 and potassium nitrate contributing 0.10 M. The enzyme solutions used in the stopped flow experiments contained potassium nitrate but no buffer, whereas the hydrogen peroxide and potassium cyanide solutions contained both potassium nitrate and bufffer. Buffer was not added to the enzyme solutions, iaa order to avoid denaturation at low pH of the enzyme prior t.o mixing of the reaction solutions. This allows measurements to be made down to about pH 2.5 where the rate of denatliratisn approaches the rate of reaction. This technique assumes that the half-life for proton equilibration is much shorter than the half-life of the reaction being studied. In the hydrogen peroxide study, the reaction mixtures contained approximately 3.5 X lW7 A4 HRP and conT N , purity umber defined as the ratio sf absorbances at 403 and 280 nm.
centrations of H2Q2varying from 7 X 10-7 !W (pH 4-10) to A4 (pH 3.4). At this concentration of HRP, the absorbaiace change during the reaction was less than 0.05 and conseyue~ltlythe approximation can be made that the absorbance change, L A , is proportionat to the photomultiplier voltage change, AV, without incurring an error of more than 5' ;. These Iow concentrations of HRP were also necessary in the pH range from 4 to 10, in order to keep the reaction slow enough to follow on the stopped flow apparatus. Chance ( I ) has shown that reaction 1 is first order in both HRP and H.02. The reaction traces were analyzed for the second-order rate constant, k,,,,,, at a particular pH value, using the following integrated second-order rate law.
in this and subsequent equaThe subscripts o, t, and tions refer to initial, intermediate, and complete stages of reaction. The initial concentration of H 2 0 2and HRP were known and the terms [N202],and [HRP], were calculated from the oscilloscope traces of photomultiplier voltage 4483 nm) using the following equations:
where V refers to the photomultiplier voItage on an arbitrary scale. To obtain V oit was necessary to make a small extrapolation to account for the amount of reaction that occurs during the dead-time of the apparatus (3 ms). The product of reaction 1 , compound I , was stable enough on the time scale of the reaction that V , could be obtained by triggering the oscilloscope trace 1-2 s after the initial trace. It was assumed that the reaction is irreversible and that a 1 :1 stoichiometry exists between MRP and H20y. In the study of the kinetics of cyanide binding to HRP. the concentratiotas of MNOa and buffer were the same as in the hydrogen peroxide study. The HRP concentration was approximately 6 X 10-7 M and the reaction was followed at 422 nm with an absorbance change during reaction of approximately 0.07. The concentration of KCN varied from about 2 X 1 W4 i%f (pH greater than 4) to 1 0 - W 6pH 2.55) and was such as to drive the reaction to completion (86). Below pH 4.2, the association equilibrium constants for cyanide with HRP are not kimown and cannot be measured readily, due to competing denattiration of the enzyme. In this pH region, the cyanide concentration was increased to such a level as to keep the half-life of the reaction in the range from 50 to 80 ms. The reaction traces showed the same overall voltage change over the entire pH range of the study, indicating that errors due to possible lack of complete complexing at low pH were minimal. In all cases, the concentration of cyanide was at least 100-fold that of the enzyme, and consequently the reactions followed first-order kinetics. The reaction traces were analyzed according to the first-order integrated rate equation with photomultiplier voltage changes converted
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DOLMAN ET AL.: HORSERADISH PEROXIBASE WITH HYDROGEN PEROXIDE
BUFFERS 0
cescodyBic acid -sodium cacodylate
KH2P04 - Na2HP04 A
boric acid
- sodium borate
s citric acid- sodium citsate
3
5
7
PH
Fa@. 1. Semilogarithmic plot of k,,, vs. pH for the reaction of WRP with H202.The curve is that one predicted by Eq. 9 using the foiliowing values: k l , 1.78 >< 10: M-I s-l; Ki, 5.64 X 10-r; K?, 1.29 X los4. Each point represents the average of three determinations of k,,, made using the same reactant solutions.
to absorbance changes before analysis of the data. The second-order association rate constant, k,,,, at a particuIar pH was calculated from the observed first-order rate constant, k,,,, and the total cyanide concentration, [CN],. using Eq. 8.
PI
k,,,
=
k,,,/ K N I T
Below pH 4, the cyanide concentrations used were large enougll to overcome the buffering capacity of the solutions, and consequently the cyanide was neutralized by adding an amount of nitric acid equivalent to the amount of KCN in solution. Bn addition to the rapid first-order reaction, corresponding to cyanide binding to HRP and with half-life ranging from 50 to 130 ms depending on reaction conditions, there was also observed a mucla slower reaction of half-life about 4 s at pH values greater than 4. This slow reaction exhibits a much smaller absorbance change than the more rapid reaction, and was not observed in the original study (16) carried out using lower cyanide concentrations. It was not studied further. The pH measurements were made with an Orion model 801 digital pH meter with a Fisher universal glass electrode and calomel electrode. Fisher certified buffer solutions were used for calibrating the electrodes. The pH values of the reaction mixtures were not measured directly but by measuring the pH of solutions made from the same K N 0 3 and bufler stock solutions, and with the same dilutions as the reaction mixtures but lacking in reactants.
The accuracy of the rate constants, k,,,,,, obtained for the reaction s f hydrogen peroxide with horseradish peroxidase is limited due to a
number of factors. including the uncertaintv in the molar of HkP(91, of dilute hydrogen peroxide solutions, and the approximation regarding absorbance and photomultiplier voltage mentioned above. As a result, the rate constants are probably uncertain to within 25% or possibly more. his large uncertainty, however, presents no real problem since kinetically important ionizable groups generally produce large changes in rate constants. The second-order rate constants, k,,,,, for reaction of HRP with H2Q2 obtained over the pH range of 2.63-9.37 are plotted sernilogarithmically in Fig. 1. Chance and coworkers (19) have previously measured the rate constant for the reaction of HRP with H 2 0 2and obtained a value of 1.15 X B07M-hs-' at an unspecified but presumably neutral pH. This compares with a value around 1.8 X 107M-I s-I at neutral pH obtained in this work. At the outset of this work, it had been hoped to make rate measurements up to a pH of at least 11, a region in which large changes occur in the spectral (17, 20), kinetic (16), and magnetic (21) properties of the enzyme. These changes have been assumed to result from ionization s f a water molecule exhibiting a p& of 18.8 (16) occupying one of the two axial coordination sites of the ferric ion. It was found, however, that when sate
498
CAN. 9.
measurements were attem~ted above DH 9.5 that the reaction traces became complex and could not be fitted by a second-order rate equation. The reaction traces exhibited an initial rapid voltage change, the amplitude of which increased with increasing pH, followed by a slower voltage change. Investigation of the reaction of the free enzyme with bufler or hydroxide solutions of pH greater than 10, in the absence of hydrogen peroxide, showed that the initial rapid reaction was caused by the pH change alone and was not due to reaction with hydrogen peroxide. As a result of this complication, no measurements of the rate of reaction 1 were made above pH 9.5. It is apparent from the plot of k,,,, vs. pH in Fig. B that ionizations are occurring in the pH range from 2.5 to 4.0 that affect the rate of reaction. Furthermore, since the slope of a plot of log k,,,, vs. pH is greater than H at low pH, there must be at least two ionizable groups involved. The simplest scheme which will account for the data plotted in Fig. B is shown below in Scheme 1.
VOL. 53, 1975
The data were analyzed for the best values of kl, K I , and K2 by the method of least mean squares using Eq. 9. In the analysis, the deviations were weighted by the factor 1 /k,,,,, in order to give equal weight to each data point. The values obtained are: for kr, 1.78 X 107M-I s-I; for KI, 5.64 X 10-2 and for K2, 1.29 X The line drawn through the data in Fig. B is obtained from Eq. 9 using these values of the parameters. A scheme kinetically equivalent to Scheme 1 involving reaction of EH with H02- can be eliminated because it leads to a rate constant for this reaction far in excess of the diflusion controlled 1imit . The results of the study of the binding of cyanide to horseradish peroxidase are plotted semilogarithmically in Fig. 2. Also indicated in Fig. 2 are the results of the previous study by Ellis and Dunford (16). There is a discrepancy between the two sets of results which cannot be accounted for. Tt should be noted in this connection that the two studies were carried out under different conditions. In this study, large concentrations of cyanide were used to drive the reaction to completion, whereas in the previous study, lower concentrations of cyanide were used and produced an equilibrium mixture containing appreciable amounts of uncomplexed as well as complexed enzyme. The previous work was also carried out on an enzyme preparation that was less pure (PN 0.9) than the present. The previous study (16) showed that in the reaction of cyanide with HWP there are three In Scheme 1, protons have been left out for kinetically important ionizations on the enzyme simplicity, E, EH1, and EKE2 refer to successively with pK, values of 10.8, 6.4, and 4.1. The protonated species of the enzyme, the ionization ionization with pK, of 6.4 has very little effect on equilibria of which are related by the ionization the rate of the reaction. The present study was constants Kg and KZ;kl is the pH independent confined to the pH region 2.55-5.5 where the rate constant for the reaction of E with H,O,. effects of the ionizations with pK, values of 6.4 The rate constants for the reactions of HzOZ and 10.8 make no important contribution to the with EHI and EH2 are assumed to be effectively observed kinetics and, as a consequence, analysis zero, and equilibration among the protonated of the results obtained in this work does not take species of enzyme is assumed to be maintained into account these ionizations. As with the results of the reaction of hydrogen throughout the reaction. This scheme leads to peroxide with MWP, the results of the reaction sf Eq. 9 for kaPp4. cvanide with WRP indicate at least two isnimble k~ boups influencing the kinetics below pH 4. [g] kapp = Reaction Scheme 2, which is directly analogous (1 4- [H+]/Kz [H+]"KIKz) to Scheme 1, can be used to account for the 4Activity coefficients have been neglected in deriving observed ka,, values for cyanide binding. %heme 2 leads t o Eq. 9 for k,,,, and this equation was Eq. 9 and in calculating [H+] from measured p~ values. I
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BHOCHEM.
+
1
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DOLMAN EY AL.: HORSERADISH PEROXIBASE W I T H HYDROGEN PEROXIDE
this study
Ellis and Dunford (16)
FIG. 2. Semilogarithmic plot of k,,, vs. pH for the reaction of WRP with WCN.The curve is that one predicted by The points for this study Eq. 9 using the following values: k l , 1.19 X lo6M-I s-l; K 1 , 1.24 X Kz, 1.10 >< are the average of two determinations made using the same reactant solutions, carried out in citric acid - sodium eitrate buffers except for the point at pH 5.50 where caeodylic acid - sodium cacodylate was used.
used to analyze for the values of kl, K1, and K2 trolled rate constant expected for a reaction in the same manner as before. between a protein and a small n~olecule(22) and must be regarded as an unlikely alternative. In this regard, Erman, in studying the binding of fluoride (23) and cyanide (24) to cytochrome c peroxidase (CcP), has argued that the neutral forms of these weakly basic ligands bind to the heme iron in peroxidases. In fact, the rate constants for the binding of HCN to the unprotonated forms of the two enzymes are the same, 1.2 X 105M-I s-I for HRB and I. 1 X 105 M-I s-I for CcP (24). The results of the analyses of the two studies The best values obtained from the analysis of are collected in Table I. Of particular interest are the data are: for kl, 1.19 X 105 M-I s-I; for K1, the values of K1 and K2for the ionizations on the 1.24 X 10-3; and for K2, 1.10 X The line free enzyme. The two K2 values are identical drawn through the data in Fig. 2 is obtained from within reasonable experimental error. The two Eq. 9 using these values of the parameters. K1 values differ by a factor of about two, and it is Scheme 2 is an extension of the original kinetic difficult to decide whether or not this difference scheme of Ellis and Dunford (16) in which the is significant. The precision of the two data sets, ionization of EH1 (pK, 3.9) in this study corre- as indicated by the standard deviations, is such sponds to the ionization of the group with pK, that the two K1 values are different; however, 4.1 in their study. As was the case in the original more realistic error limits are undoubtedly much study, there is a kinetically indistinguishable larger than the standard deviations. It is quite alternative mechanism involving reaction of CN- possible that there are systematic errors present with EHL.Based on a pK, value of 9.0 (16) for in either or both sets of kinetic data that could HCN at an ionic strength of 0.1 I, the rate con- account for the observed difference in K1 values. stant for this reaction is 1.3 X 1010M-' s-I. This This is particularly likely at low pH where r diffusion eon- denaturation of the enzyme is rapid. The possivalue is at the upper limit f ~ the
CAW. J. BIOCHEM, V O t . 53, 1975
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TABLE 8 . Values for the parameters of Eq. 9 obtained from analysis of the data in Figs. 1 and 2" Reaction
k l , M-l s-I
KI
HRP f Hz82 (Schetnel)
(1.78+0.$3)X107
(5.64+0.42)Xl0-"(%.29+0.08)XleB-'
HRP f HCN (Scheme 2)
(1 .19+0.02)X105
(l024kO.07)X10-"1.
Kz
10kQ.$5)X10-2
UThe errors are the standard deviations determined from the least-squares analysis of the data. Actual errors are considerably larger.
bility cannot be dismissed, however, that the two Kl values refer to two distinct ionizable groups. -
Discussion Comparison of the kinetic results over the pH range of 2.5-9.5 for the reactions of H202 and HCN with HRP indicates that the a ~ ~ a r erate nt constants for the two reactions are affected in the same manner by ionizations of specific groups on the enzyme. Both reactions reveal the presence of two kinetically important ionizations on the enzyme with pKLLvalues in the range of 2.9-4.0. The HCN-HRP kinetic results reveal, in addition, a group on the enzyme with a pKa value of 6.4 (16) that is not evident in the H202-HRP reaction, Ionization of this group with pK:, of 6.4 has little eKect on the kinetics of HCN binding to HRP and it was included primarily to satisfy the principle of detailed balancing (16). The similarity of the two reactions in regard to the effect of pH on the kinetics suggests that there are similarities in their mechanisms. A possible explanation for this similarity might be found in the following mechanism (Scheme 3) for reaction of H 2 0 2with HRP.
tional assumption that the concentration of intermediate remains low relative to the concentration of free enzyme, results in Eq. 18 for the rate of compound I formation.
6 101
d[compound I] dt
I P
compound I SCHEME 3
In Scheme 3, E-Fe(H20) indicates the free enzyme with a water molecule coordinated to the ferric ion at one of its axial positions, and E-Fe(H202) indicates hydrogen peroxide coordinated in the same position to the iropi atom. Application of the steady-state assumption to the intermediate E-Fe(Hn02), with the addi-
Thus the two step process leads to a prediction of second-order kinetics, as is observed for compound I fornaation, with k,,, identified with klk2 /(k-1 f k2). The first step in the mechanism outlined in Scheme 3 is analogous to the reaction of HCN with HRP and consequently Bdr in Scheme 3 should show the same pH dependence due to ionizable groups on the enzyme as is found in the HRP-HCN reaction. In turn, the pH dependence of kl, according to Eq. 10, is carried into k,,,,. A more complicated pH dependence of k,,,, than is observed is predicted by mechanism 3, however, if the two ionizable groups corresponding to K I and K2 also affect the rate of the second step, formation of compound I from E-Fe(H203). Support for the above mechanism can be found in the work of Portsmouth and Beal (25) who have found that hydrogen peroxide reacts with deuterohemin to form a product capable sf oxidizing typical peroxidase substrates. The rate of formation sf this product is not first order in the concentration of; H 2 0 2 but rather shows a saturation effect with respect to increasing H2On concentration. This result has been interpreted by Portsmouth and Beal as indicating the formation of a hemin-H202 con~plexas an intermediate en route to formation of the persxidatic product. This, s f course, is a direct analog of mechanism 3.
DOLMAN ET AL.: HORSERADISH PERCBXIDASE WITH HYDROGEN PEROXIDE
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A reasonable proposal for the identity of the pK, values of 3.2 (or 2.9) and 3.9 is that they belong to the carboxyl groups of the porphyrin propisnic acid side chains. Support for such a view is available from the work of Maehly (26) and of Tamura et aE. (27). Maehly showed that protohemin and protoporphyrin both bind to horseradish apopessxidase to produce strong Soret band absorption at 403 nrn, characteristic sf native horseradish peroxidase, whereas protohemin dimethyl ester and protoporphyrin dimethyl ester do not exhibit such adsorption changes. Tarnma el ak. also observed that the apoenzyme has a reduced aEnity for the protohemin dimethyl ester as compared with its affinity for native protohemin. They found that the enzyme reconstituted from apoenzyme and the protohemin dimethyl ester has a much reduced affinity for ligands such as cyanide and a much reduced reactivity towards hydroperoxides. Since protonation of both of the carboxyl groups of the hernin would be expected to produce effects similar to those observed irn the dimethyl ester, it seems reasonable to identify the two ionizable groups found in the MRP-H202 study with the hemin carboxyl groups. The situation is not clear cut, however, since these workers (27) found that protohemin monomethyl ester combines with apoemyrne to form an enzyme reactive towards both cyanide and hydroperoxides. If the kinetically important ionizable groups are indeed the hemin carboxyl groups then this result would lead one to expect the mornoprotonated species, EH1, to be reactive towards hydrogen peroxide, contrary to observation. It is, of course, possible that the species EH1 has substantial b i t reduced reactivity towards H202,as compared with that of E, which was not detected in this study. Although one of the ultimate goals of this type of study is to identify and to determine the role of ionizable groups that participate in the catalytic cycle of the enzyme, this goal is still far from being present no obvious identification of the pKa values 3-2 2-9) and 3-9 found on the free enzyme can be made with pK, values on compounds I and 11 of H W P round in previous siudies (8-14).
50 1
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