©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Spectral Characterization and Chemical Modification of Catalase-Peroxidase from Streptomyces sp. (*)

Hong-Duk Youn (1), Yang-In Yim (1), Kwan Kim (2), Yung Chil Hah (1), Sa-Ouk Kang (1)(§)

From the (1) Department of Microbiology, College of Natural Sciences, and Research Center for Molecular Microbiology, Seoul National University, Seoul 151-742, Republic of Korea and the (2) Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Catalase-peroxidase was purified to near homogeneity from Streptomyces sp. The enzyme was composed of two subunits with a molecular mass of 78 kDa and contained 1.05 mol of protoporphyrin IX/mol of dimeric protein. The absorption and resonance Raman spectra of the native and its cyano-enzyme were closely similar to those of other heme proteins with a histidine as the fifth ligand. However, the peak from tyrosine ring at 1612 cm, which is unique in catalases, was not found in resonance Raman spectra of catalase-peroxidase. The electron paramagnetic resonance spectrum of the native enzyme revealed uniquely two sets of rhombic signals, which were converted to a single high spin, hexacoordinate species after the addition of sodium formate. Cyanide bound to the sixth coordination position of the heme iron, thereby converting the enzyme to a low spin, hexacoordinate species. The time-dependent inactivation of the enzyme with diethyl pyrocarbonate and its kinetic analysis strongly suggested the occurrence of histidine residue. From the above-mentioned spectroscopic results and chemical modification, it was deduced that the native enzyme is predominantly in the high spin, ferric form and has a histidine as the fifth ligand.


INTRODUCTION

Cellular metabolism of molecular oxygen results in inadvertant generation of reactive oxygen species such as superoxide anion radical, hydroxyl radical, and hydrogen peroxide, and their cytotoxic effects are removed by specific enzyme systems (1) . Two classes of heme proteins designated hydroperoxidases, that is, catalase and peroxidase, are involved in the neutralization of hydrogen peroxide. The former is usually defined as an enzyme catalyzing the dismutation of HO to O and HO, whereas the latter uses HO to oxidize a variety of compounds (2, 3) . The catalases isolated from animals (2, 4) , plant (5) , and microorganisms (6, 7, 8) are similar to each other; they are composed of four identical subunits with a native molecular mass in the range of 225-270 kDa, contain one protoporphyrin IX group/subunit, exhibit a broad pH range of 5-10. They are inhibited by 3-amino-1,2,4-triazole (2, 9) and are not reduced by dithionite (3) . Most of the peroxidases, on the other hand, are composed of one subunit showing variety in molecular mass and are reduced by dithionite (3) . In particular, it has been reported that fungal peroxidases, such as peroxidase from Pleurotus ostreatus(10) , lignin peroxidase (11), or manganese peroxidase (12) from Phanerochaete chrysosporium, were excreted to culture fluid, and they were glycosylated in the range of 20-40% (10, 11, 12) .

A novel type of hydroperoxidase, so-called catalase-peroxidase has been recently proposed to exhibit both catalatic and peroxidatic activities (9). This enzyme is distinct from typical catalases and peroxidases, with respect to the fact that this enzyme is reduced by dithionite like peroxidase (3) , not inhibited by 3-amino-1,2,4-triazole (2) , inactivated by hydrogen peroxide, and possesses narrow pH range for its maximal activity (9) . Catalase-peroxidases have been found in prokaryotes such as Escherichia coli(13) , Salmonella typhimurium(14) , Klebsiella pneumoniae(15) , Halobacterium halobium(16) , and Rhodopseudomonas capsulata(17) . Catalase-peroxidases are considered to be ancestral forms of catalase or peroxidase in evolutionary flow (9) . It has been proposed that they may act as catalases because the substrates for peroxidatic activity are not found in cytoplasm (13) .

A tyrosine residue was known to be the fifth ligand to the heme iron in case of catalases, as determined by the crystallographic (18) and resonance Raman spectroscopic studies (19) , a histidine residue as the fifth ligand in case of peroxidases such as horseradish peroxidase (20), lignin peroxidase (21) , and manganese peroxidase (22) , and a cysteine residue as the fifth ligand in case of cytochrome P450 (23, 24) . However, the structural information of the heme environment of catalase-peroxidase has not been clarified yet.

To understand the bifunctional mechanism of catalase-peroxidase, in the present paper we report the structural information of catalase-peroxidase purified from Streptomyces sp. IMSNU-1 by absorption, resonance Raman and electron paramagnetic resonance spectroscopic analyses, and chemical modification.


EXPERIMENTAL PROCEDURES

Materials

Dimethyl suberimidate, diethyl pyrocarbonate, o-dianisidine, DEAE-Sephacel, Superose 12, and phenyl-Sepharose were purchased from Sigma; hydrogen peroxide was from Merck; Protein-Pak DEAE 5PW was from Waters; and molecular mass markers for gel filtration and SDS-PAGE() from Boehringer Mannheim. All other reagents used were of the highest quality generally available.

Microorganism and Growth Conditions

Streptomyces sp. IMSNU-1, isolated from the Kwanak mountain in Seoul, was maintained on Bennett medium. The spores of Streptomyces sp. IMSNU-1 were collected with 20% glycerol from Bennett medium grown at 28 °C for 5 days. 100 µl of spore suspension with the concentration of 4 10/ml was inoculated into 200 ml of modified YEME medium (0.5% yeast extract, 0.5% casamino acid, 0.5% peptone, 0.3% malt extract, and 1% dextrose, pH 7.0). The microorganism was cultivated aerobically in 500-ml baffled flasks at 28 °C for 48 h.

Enzyme Assays

Peroxidase activity was assayed using 0.5 mMo-dianisidine as a substrate in the presence of 4 mM HO in 50 mM sodium acetate buffer, pH 5.5. One unit of peroxidase activity was defined as the amount of enzyme that converted 1 µmol of o-dianisidine/min ( = 11,300 M cm) (13) .

Catalase activity was determined spectrophotometrically by measuring the decrease in the A using 20 mM HO in 50 mM sodium phosphate buffer, pH 6.0. One unit of catalase activity was defined as the disappearance of 1 µmol of HO/min ( = 43.6 M cm) (25) . Catalase activity was stained on polyacrylamide gel as described previously (26) .

Enzyme Purification

The grown mycelia were collected by aspiration on filter paper in a Büchner funnel and washed with 0.85% KCl solution. The washed mycelia were suspended in buffer A (20 mM sodium phosphate, pH 7.4, containing 0.1 mM EDTA), and broken with a bead beater (Biospec Productions, Inc., U. S. A.) for 5 min, and the homogenate was centrifuged (4000 g for 15 min). Solid ammonium sulfate was added to the supernatant up to 80% saturation. The precipitate was collected by centrifugation (15,000 g for 20 min) and resuspended in buffer A. The resuspended solution was dialyzed against buffer A overnight. The dialyzate was loaded onto a DEAE-Sephacel (4 40 cm) previously equilibrated with buffer A, and the proteins were eluted with a linear gradient of 0-0.5 M NaCl in buffer A. The enzyme was pooled and exchanged with buffer B (20 mM sodium phosphate, pH 6.0) using an Amicon YM10 membrane. The pooled active fractions were purified by Waters Delta Prep 4000 chromatography system using a Protein-Pak DEAE 5PW column (2.15 15 cm) equilibrated with buffer B. After the column was washed with 0.2 M NaCl in buffer B, proteins were eluted with a linear gradient of 0.2-0.35 M NaCl in buffer B. The active fractions were pooled and exchanged with buffer C (20 mM sodium phosphate, pH 6.8, containing 0.5 M ammonium sulfate) using an Amicon YM10 membrane. After the pooled active fractions were loaded onto a phenyl-Sepharose (1.2 10 cm) equilibrated with buffer C, the proteins were eluted with a reversely linear gradient of 0-0.5 M ammonium sulfate in 20 mM sodium phosphate, pH 6.8. The active fractions were collected and exchanged with 50 mM sodium phosphate, pH 6.0, and stored at -70 °C. The purity of enzyme was estimated using a Spectroscan XL densitometer (Pharmacia Biotech Inc.).

Molecular Mass Determination

The molecular mass of the purified enzyme was determined by the gel filtration chromatography on Superose 12 (2 60 cm), calibrated with catalase (240 kDa), aldolase (158 kDa), albumin (68 kDa), and chymotrypsinogen A (25 kDa). For cross-linked proteins, dimethyl suberimidate was used as a cross-linking agent (27) , and cross-linked proteins were electrophoresed on 5% SDS-PAGE (28) . The gel was calibrated with -macroglobulin (170 kDa), -galactosidase (116 kDa), fructose-6-phosphate kinase (85 kDa), and glutamate dehydrogenase (55 kDa).

Spectroscopic Measurements

The absorption spectra of all samples were measured by means of a Shimadzu UV-265 spectrophotometer. Spectra were obtained at 25 °C using a 1-cm quartz cuvette containing native and adductive enzyme dissolved in 50 mM sodium phosphate buffer, pH 6.0. The heme content of the enzyme was determined by measuring the absorbance of heme-pyridine complex at 556 nm ( = 33,200 M cm) (29) .

The high frequency (>1100 cm) resonance Raman (rR) spectra of native and cyano-adduct were recorded with a Spex model 1877E triple spectrometer and obtained at room temperature with 90° scattering geometry. Excitation was provided by a Spectra Physics 164-05 argon ion laser. Multiple scans were added to improve the signal to noise ratio of spectra.

The EPR spectra were measured by means of a Bruker ESP 300S EPR spectrometer (Germany). Probe temperature was regulated with a helium cryostat equipped with a temperature control unit.

Chemical Modification of Catalase-Peroxidase with Diethyl Pyrocarbonate

Stock solution was made by the dilution of diethyl pyrocarbonate into absolute ethanol. The concentration of the diethyl pyrocarbonate solution was measured by the absorbance at 230 nm ( = 3000 M cm) by the addition of ethanolic stock solution of diethyl pyrocarbonate to 10 mM imidazole-HCl buffer, pH 7.5 (30) . The quantitation of the formation of N-carbethoxyhistidine residues was determined by the increase of the absorbance at 242 nm ( = 3200 M cm) (31) .


RESULTS AND DISCUSSION

Purification of Catalase-Peroxidase

The enzyme was purified 34.2-fold relative to the cell extract with a recovery of 21.6%, as shown in . The final preparation gave a major single band with both catalatic and peroxidatic activity on 7.5% nondenaturing polyacrylamide gel (Fig. 1A). The final preparation contained minor contaminated proteins on silver-stained SDS-polyacrylamide gel (Fig. 1B) (32) , and the purity of the enzyme was estimated to be 86% by means of densitometry.


Figure 1: PAGE of catalase-peroxidase from Streptomyces sp. IMSNU-1. A, 7.5% nondenaturing polyacrylamide gel: lane a, peroxidase activity was stained using o-dianisidine and HO as substrates as described in Ref. 13; lane b, catalase activity was stained as described in Ref. 26; lane c, protein was stained with Coomassie Blue. 3 µg of each protein was loaded into each lane. B, 5% SDS-PAGE: lane a, native enzyme (3 µg); lane b, cross-linked protein (3 µg) treated with dimethyl suberimidate as described in Ref. 27. The gel was calibrated with -macroglobulin (170 kDa), -galactosidase (116 kDa), fructose-6-phosphate kinase (85 kDa), and glutamate dehydrogenase (55 kDa).



Molecular Properties

The molecular mass of the enzyme was determined to be 78 kDa after SDS-PAGE in the presence or absence of 2-mercaptoethanol. To obtain an insight into the subunit composition, catalase-peroxidase was incubated with a cross-linking agent, dimethyl suberimidate, prior to denaturation and electrophoresis. Two bands (78 and 157 kDa) were visualized after silver staining (Fig. 1B). The native enzyme has a molecular mass of 160 kDa as estimated by Superose 12 gel filtration chromatography (2 60 cm). This enzyme is, therefore, considered to be composed of two subunits that are not covalently cross-linked.

Absorption Spectra of Catalase-peroxidase

The absorption spectra of the native enzyme exhibited maxima at 407, 500, and 635 nm, and the ratio of A/A reached 0.54 (Fig. 2A). The molar absorption coefficient of the enzyme was 107.03 mM cm at 407 nm. The treatment of the enzyme with pyridine-NaOH-dithionite, customarily used to determine pyridine hemochrome (29) , gave a typical spectrum of iron protoporphyrin IX, whose absorption bands appeared at 418, 525, and 556 nm. On the basis of the molar absorption coefficient of 33,200 M cm at 556 nm for the pyridine hemochrome (29) , the heme content was calculated to be 1.05 mol of heme/mol of dimeric protein. This low heme content corresponds to the A/A ratio of 0.54. It is not likely that this is due to loss of heme during purification procedure, because the mobility of this enzyme on polyacrylamide gel was not modified during purification. It has been reported that catalase-peroxidases found in other bacterial systems have low heme content. The enzyme of E. coli HP-1 had 2.07 mol of heme/mol of tetrameric protein, that of H. halobium had 1.43 mol of heme/tetramer, and that of R. capsulata had 1-1.2 mol of heme/tetramer (13, 16, 17) . The reduction of the enzyme with dithionite decreased the Soret band and shifted it to 437 nm, and a new peak appeared at 559 nm as the peaks at 550 and 638 nm decreased (Fig. 2A). The addition of KCN to the enzyme shifted the Soret band to 422 nm, and a new peak appeared at 538 nm (Fig. 2B).


Figure 2: Absorption spectra of catalase-peroxidase. A, enzyme (0.24 µM) was dissolved in 50 mM sodium phosphate buffer, pH 6.0. The reduced form (- - -) was obtained by the addition of 1 mM sodium dithionite to the native form (--). Inset shows the pyridine hemochrome spectrum of catalase-peroxidase. B, enzyme (0.15 µM) was dissolved in 50 mM sodium phosphate buffer, pH 6.0. The cyano-adduct (- - -) was obtained by the addition of 30 mM KCN to the native enzyme (--).



The absorption bands of the native enzyme and its CN-ligated form are summarized in along with the data from a variety of other heme proteins. The absorption maxima of catalase-peroxidase at 407, 500, and 638 nm are similar to those of high spin heme proteins with a histidine as the fifth ligand (20, 21, 22, 33) but very different from that of cytochrome P450 (391, 500, and 646 nm) with a cysteine as the fifth ligand (23) as well as that of cytochrome b, low spin, hexacoordinate species (34). The absorption maxima of CN-adduct of catalase-peroxidase are virtually identical to those of the low spin, hexacoordinate CN-adducts of horseradish peroxidase (20) , lignin peroxidase (21) , manganese peroxidase (22) , and metMb (33) , but distinct from that of the low spin, hexacoordinate CN-adduct of cytochrome P450 (). These absorption patterns suggest that the fifth ligand to the heme iron of catalase-peroxidase can be a histidine.

rR Spectra of Catalase-Peroxidase

High frequency (1100-1700 cm) rR spectra of the native enzyme and its CN-ligated form obtained with the excitations at 488.0 and 514.5 nm are shown in Fig. 3. The dominant rR bands in high frequency region are the oxidation state, spin state, and coordination number marker bands (23, 35) . The frequency marker of the most characteristic oxidation state is found at 1368-1376 cm for ferric hemes. The position of is thought to reflect the electron population in the porphyrin * orbitals, with an increasing population weakening the porphyrin bonds and bringing about the decrease in vibrational frequency (36, 37, 38) . The frequencies of the most characteristic spin-states are , , and . The vibrational energies of these motions are sensitive to the spin state of heme iron and correlated to the expansion of the porphyrin core (39, 40) . For ferric hemes, the observed ranges for the markers of these high spin and low spin are as follows: for (polarized, maximal intensity with Soret excitation), 1478-1494 cm (high spin) versus 1497-1508 cm (low spin); for (depolarized, maximal intensity with and excitation), 1605-1626 cm (high spin) versus 1636-1642 cm (low spin); for (anomalously polarized, maximal enhancement with and excitation), 1548-1576 cm (high spin) versus 1577-1590 cm (low spin). The markers of three spin states are also sensitive to the coordination number of the heme (23, 35) . Band appears at 1605-1612 cm for high spin ferric hexacoordinate but at 1622-1626 cm for high spin ferric pentacoordinate, while band lies at 1478-1482 cm for hexacoordinate but at 1487-1494 cm for pentacoordinate. On the other hand, band lies at 1548-1562 cm for hexacoordinate but at 1565-1576 cm for pentacoordinate.


Figure 3: Resonance Raman spectra of native and cyano-ligated catalase-peroxidase. A, enzyme was concentrated to 120 µM in 50 mM sodium phosphate buffer, pH 6.0. B, cyano-adduct formed by the addition of 30 mM KCN to the sample in A. Upper spectra (a) of each panel were obtained after the excitation of 488.0 nm with 75 milliwatts and lower spectra (b) after the excitation of 514.5 nm with 120 milliwatts. All spectra were obtained at room temperature with 90 ° scattering geometry.



Upon excitation in the visible range at 488.0 and 514.5 nm, the native catalase-peroxidase gives a typical rR spectrum of high spin ferric heme (Fig. 3) with , , , and at 1372, 1490, 1622, and 1557 cm, respectively. The rR spectra of three catalases from bovine liver, Aspergillus niger, and Micrococcus luteus display a peak at 1612 cm, which is a distinct feature with 488.0 nm excitation but diminishes to a shoulder upon excitation at 514.5 nm (19) . Tyrosine ligation in high spin pentacoordinate heme is most readily identified by a polarized rR band at 1612 cm maximally enhanced with visible excitation (19). But this band, the characteristic of tyrosine ligation, was not found in the rR spectra of catalase-peroxidase (Fig. 3A).

The ligation of CN to the native enzyme causes the formation of low spin hexacoordinate ferric heme (Fig. 3B and I). Band of 1622 cm in the spectra of the native form is shifted to 1636 cm in those of CN-adduct. Band and are shifted from 1490 cm and 1557 cm to 1500 cm and 1580 cm, respectively. These spectral shifts correspond to those for the conversion of high spin ferric heme to low spin ferric heme.

The rR spectra of CN-adduct are fairly similar to that of CN-lignin peroxidase (41) . However, these are somewhat different from those of CN-horseradish peroxidase (20) , and CN-cytochrome c peroxidase (42) (I).

EPR Spectra of Catalase-Peroxidase

EPR spectrum of catalase-peroxidase, as shown in Fig. 4A, is composed of two sets of rhombic signals having resonances at g = 6.619, 5.597, and 2.041, and g = 6.021, 5.117, and 2.041. The native enzyme treated with 0.2 M sodium formate, pH 7.0, is converted to a major single rhombic high spin species with resonances at g = 6.021, 5.117, and 2.041 (Fig. 4B). The g values for catalase-peroxidase are compared with those for other heme proteins (). The g values for the native enzyme are distinct from those for other heme proteins, in particular, those for cytochrome P450 (). It has been reported that commercial preparations of catalase from bovine liver (2) and that of Neurospora crassa(44) contain ferric hemes in two different symmetry forms.


Figure 4: EPR spectra of catalase-peroxidase. A, native enzyme (19.1 µM) was dissolved in 50 mM sodium phosphate buffer, pH 6.0. B, sample treated with 0.2 M sodium formate, pH 7.0. C, cyano-ligated form by the addition of 30 mM KCN to the sample in A. Conditions in A and B: microwave frequency, 9.49 GHz; microwave power, 20 milliwatts, modulation frequency, 100 kHz; modulation amplitude, 10 G; sweep width, 6000 G; conversion, 81.92 ms. Conditions in C: microwave frequency, 9.41 GHz; microwave power, 2 milliwatts; modulation frequency, 100 kHz; modulation amplitude, 10 G; sweep width, 4400 G; conversion, 40.96 ms.



Furthermore, EPR spectrum of CN-adduct shows a single rhombic low spin state having resonances at g = 3.147 and 2.186 (Fig. 4C), which is similar to that for CN-lignin peroxidase (42) but is distinct from that for CN-catalase (44, 45) or CN-cytochrome P450 (24) . A small amount of high spin nonheme iron appears to be present as judged by the signal near g = 4.3.

The presence of one rhombic EPR signal for either high spin ferric heme-formate or low spin ferric heme-cyanide suggests that the native enzyme contains only one type of heme group.

Chemical Modification of Catalase-peroxidase with Diethyl Pyrocarbonate

Diethyl pyrocarbonate has been used for the selective modification of histidine residues (31) . Catalase-peroxidase was inactivated in a time-dependent manner when incubated with various concentrations of diethyl pyrocarbonate at pH 6.5 (Fig. 5A), k values from each slope of the curves in A were proportional to the concentration of diethyl pyrocarbonate and the slope of the curve yielded the second-order rate constant, which was calculated to be 0.095 Mmin (Fig. 5B). The linearity means that the modification is a simple bimolecular reaction between enzyme and diethyl pyrocarbonate. As shown in Fig. 6, the ultraviolet difference spectrum of the modification of catalase-peroxidase with diethyl pyrocarbonate demonstrated an increase in absorbance at 242 nm, indicating the N-carbethoxylation of histidine residues. The number of histidine residues modified upon incubation of 10 µM catalase-peroxidase with 4 mM diethyl pyrocarbonate (400-fold excess reagent) was calculated to be 4.2, which suggests that there is a population of nonessential histidine residues.


Figure 5: The inactivation of catalase-peroxidase with diethyl pyrocarbonate. Catalase-peroxidase (10 µM) was incubated with various concentrations of diethyl pyrocarbonate at pH 6.5 and 25 °C. A, determination of pseudo first-order rate constant of inactivation. Data was plotted to -ln(A/A) = kxt, and the curves were fit by least square analysis. B, determination of the second-order rate constant of inactivation. The slope of this curve is equal to the second-order rate constant, which is calculated to be 0.095 min at pH 6.5 and 25 °C.




Figure 6: The ultraviolet difference spectrum of the modification catalase-peroxidase with diethyl pyrocarbonate. Catalase-peroxidase (10 µM) was incubated with 0.4 mM diethyl pyrocarbonate at pH 6.5. Scans were repeatedly collected over every 2-min periods.



A kinetic study was performed to determine the number of essential histidine residues in catalase-peroxidase (47) . The inactivation of enzyme with diethyl pyrocarbonate can be described in the following expression.

On-line formulae not verified for accuracy

The reciprocal of the half-life time (t) is substituted for k, which has no effect on the slope (47) . The slope of the plot is near unity (n = 1.73 ± 0.08) per mol of dimer (Fig. 7). This indicates that the inactivation of enzyme with diethyl pyrocarbonate involves the modification of about one essential residue/subunit of enzyme since catalase-peroxidase is composed of two subunits.


Figure 7: The apparent order of the inactivation of catalase-peroxidase. The slope of this line is 1.73/mol of dimeric protein. Since catalase-peroxidase is composed of two subunits, the modification of one essential histidine is required to cause inactivation of one subunit of catalase-peroxidase.



In addition to histidine, it has been previously shown that diethyl pyrocarbonate can react with cysteine, tyrosine, and lysine residues (48). The formation of O-carbethoxylation of tyrosine residue is monitored by a dramatic absorbance decrease at 278 nm ( = 13,101 M cm) (49) . Tyrosine residue is not involved in the modification of the enzyme with diethyl pyrocarbonate since there is little change in absorbance at 278 nm. The formation of N-carbethoxylation of lysine residue is distinguished from that with histidine residue; N-carbethoxylation of histidine residue is reversible when treated with hydroxylamine, while that of lysine residue is irreversible (50) . The absorbance at 242 nm decreased when hydroxylamine was added to catalase-peroxidase treated previously with diethyl pyrocarbonate, indicating that N-carbethoxylation of histidine residue caused the inactivation of enzyme (Fig. 8). Cytochrome P450, of which the fifth ligand has been known to be cysteine, is converted to cytochrome P420 by the modification with diethyl pyrocarbonate (51) . However, the addition of diethyl pyrocarbonate to catalase-peroxidase had no effect on the Soret band, indicating that diethyl pyrocarbonate is not attributed to the formation of S-carbethoxylation of cysteine.


Figure 8: The effect of hydroxylamine on the rate of inactivation of catalase-peroxidase. Catalase-peroxidase (10 µM) was incubated with 0.4 mM diethyl pyrocarbonate at pH 6.5 and 25 °C, after 8 min, 0.05 mmol of hydroxylamine was added to the mixture. The change in absorbance at 242 nm was monitored.



Diethyl pyrocarbonate reacts with the unprotonated amino acid residues, and the pH dependence of the inactivation process provides information about the identity of the modified residue (52) . Pseudo first-order rate constant was determined and plotted versus pH (Fig. 9A). The experimental values can be replotted according to the following equation

On-line formulae not verified for accuracy

where K is the dissociation constant of a reacting group and (k) is the pseudo first-order rate constant of the unprotonated reacting group (53). From Fig. 9B, (k) was calculated to be 0.098 min at x-intercept, and the slope was used to calculate the apparent pK value of 6.1, which suggests that the inactivation of catalase-peroxidase with diethyl pyrocarbonate is due to the modification of histidine residue.


Figure 9: The effect of pH on the rate of inactivation of catalase-peroxidase. Catalase-peroxidase (8 µM) was incubated with 0.8 mM diethyl pyrocarbonate at various pH values. A, pseudo first-order rate constants were plotted versus pH. B, the experimentally determined pseudo first-order rate constants were plotted according to Equation 2. The slope and the x-intercept correspond to a k and (k) of 8.92 10 (pK of 6.1) and 0.098 min, respectively.



  
Table: Purification step of catalase-peroxidase from Streptomyces sp. IMSNU-1


  
Table: Absorption spectral maxima of Streptomyces catalase-peroxidase (StCP) and other heme proteins


  
Table: 0p4in Ref. 42.(119)

  
Table: Comparison of EPR g values for Streptomyces catalase-peroxidase (StCP) and other heme proteins



FOOTNOTES

*
This work was supported by the Korea Science and Engineering Foundation research grant for SRC (Research Center for Molecular Microbiology, Seoul National University). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 82-2-880-6703; Fax: 82-2-888-4911; E-mail: kangsaou@alliant.snu.ac.kr.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; CytP450, the camphor-hydroxylating cytochrome P450 from Pseudomonas putida; EPR, electron paramagnetic resonance; rR, resonance Raman.


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