From the
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
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
H
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.
Catalase activity was determined spectrophotometrically by measuring
the decrease in the A
The high frequency (>1100 cm
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.
The ligation of CN to the native enzyme causes the formation of low
spin hexacoordinate ferric heme (Fig. 3B and
I). Band
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).
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.
On-line formulae not verified for accuracy
The reciprocal of the half-life time (t) is substituted
for k
On-line formulae not verified for accuracy where K
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.
O
to O
and H
O, whereas
the latter uses H
O
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) .
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) .
using 20 mM
H
O
in 50 mM sodium phosphate buffer,
pH 6.0. One unit of catalase activity was defined as the disappearance
of 1 µmol of H
O
/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) .
) 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.
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) .
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).
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.
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.
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 M
min
(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) =
k
xt, 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.
, 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
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:
Comparison of EPR
g values for Streptomyces catalase-peroxidase (StCP) and other heme
proteins
, the camphor-hydroxylating cytochrome P450 from
Pseudomonas putida; EPR, electron paramagnetic resonance; rR,
resonance Raman.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.