(Received for publication, July 13, 1995; and in revised form, September 11, 1995)
From the
The oxidation of lipoproteins is considered to play a key role
in atherogenesis, and tyrosyl radicals have been implicated in the
oxidation reaction. Tyrosyl radicals are generated in a system
containing myeloperoxidase, HO
, and tyrosine,
but details of this enzyme-catalyzed reaction have not been explored.
We have performed transient spectral and kinetic measurements to study
the oxidation of tyrosine by the myeloperoxidase intermediates,
compounds I and II, using both sequential mixing and single-mixing
stopped-flow techniques. The one-electron reduction of compound I to
compound II by tyrosine has a second order rate constant of (7.7
± 0.1)
10
M
s
. Compound II is then reduced by tyrosine to native
enzyme with a second order rate constant of (1.57 ± 0.06)
10
M
s
. Our study further revealed that, compared with
horseradish peroxidase, thyroid peroxidase, and lactoperoxidase,
myeloperoxidase is the most efficient catalyst of tyrosine oxidation at
physiological pH. The second order rate constant for the
myeloperoxidase compound I reaction with tyrosine is comparable with
that of its compound I reaction with chloride: (4.7 ± 0.1)
10
M
s
. Thus, although chloride is considered the major
myeloperoxidase substrate, tyrosine is able to compete effectively for
compound I. Steady state inhibition studies demonstrate that chloride
binds very weakly to the tyrosine binding site of the enzyme. Coupling
of tyrosyl radicals yields dityrosine, a highly fluorescent stable
compound that had been identified as a possible marker for lipoprotein
oxidation. We present spectral and kinetic data showing that dityrosine
is further oxidized by both myeloperoxidase compounds I and II. The
second order rate constants we determined for dityrosine oxidation are
(1.12 ± 0.01)
10
M
s
for compound I and
(7.5 ± 0.3)
10
M
s
for compound II. Therefore, caution must be
exercised when using dityrosine as a quantitative index of lipoprotein
oxidation, particularly in the presence of myeloperoxidase and
H
O
.
A major risk factor for coronary artery disease is an elevated
level of low density lipoproteins (LDL), ()the major carrier
of blood cholesterol(1) . Thus, the reduction of LDL levels has
been a prominent preventive measure against atherosclerosis. However,
evidence has accumulated indicating that oxidized LDL rather than
native LDL triggers the pathological events leading to
atherosclerosis(1, 2, 3, 4) . Foam
cells or lipid-laden macrophages, derived from circulating monocytes,
are the earliest cellular components of atherosclerotic
lesions(5) . It has been found that normal macrophages in
culture cannot be converted to foam cells by simple incubation with
even very high concentrations of native LDL. It is postulated that
circulating LDL first undergo oxidative modification and then the
oxidized LDL convert normal macrophages into foam
cells(6, 7) .
Myeloperoxidase, a heme protein abundant in phagocytes(8) , is a potential physiological catalyst for lipoprotein oxidation(9, 10) . Recent studies demonstrate links between myeloperoxidase and oxidative damage to proteins and lipids(11, 12) . The identification of myeloperoxidase in human atherosclerotic lesions (13) is support for the hypothesis that myeloperoxidase is indeed involved in lipoprotein oxidation.
While myeloperoxidase follows the normal peroxidase cycle(14, 15) ,
where a single two-electron oxidation of native enzyme (MPO) to compound I (MPO-I) is followed by two successive one-electron reductions back to native enzyme via compound II (MPO-II), it shares an ability with eosinophil peroxidase (16) among the mammalian peroxidases in utilizing chloride to produce hypochlorous acid(17, 18) ,
HOCl is a potent oxidizing agent that plays a cytotoxic role against invading bacteria, viruses, and tumor cells(8) . However, it is also known to injure normal tissues by bleaching heme groups and oxidatively destroying electron transport chains (19) as well as chlorinating amino acids and unsaturated lipids(10, 20, 21) . HOCl secreted by activated neutrophils can also cause oxidation of lipoproteins in vivo(22) . Moreover, myeloperoxidase has been found to convert cholesterol to chlorohydrins and epoxides by a reaction involving HOCl(11) .
Another potential lipoprotein oxidative pathway involving myeloperoxidase implicates the tyrosyl radical produced via one-electron oxidation of tyrosine by MPO-I(12, 23) . When two tyrosyl radicals combine the major product is o,o`-dityrosine, an intensely fluorescent compound(24, 25) . Fluorescence due to dityrosine has been observed in the reaction of tyrosine with linoleic acid 13-monohydroperoxide in the presence of methemoglobin(26) . Dityrosine has also been detected in high density lipoproteins (HDL) treated with horseradish peroxidase, hydrogen peroxide, and tyrosine(27) .
The concentration of free tyrosine in human
blood plasma is 55 µM(28) and ranges from 22 to
83 µM in venous blood(29) . Since dityrosine
formation has been observed even in the presence of physiological
concentrations of chloride(12, 30) , the major MPO
substrate, it has been suggested that tyrosine can be an effective
reducing substrate for MPO. While the literature abounds with evidence
for oxidative tyrosylation of
lipoproteins(12, 23, 27, 30) ,
limited if any details are reported on the myeloperoxidase-catalyzed
one-electron oxidation of tyrosine. In this paper we report spectral
and transient kinetic data for the oxidation of tyrosine by the
MPO/HO
system. Rate constant determination for
MPO-I reactions were made possible by employing sequential mixing
stopped-flow techniques. We also report rate constants for tyrosine
oxidation by MPO-II. The contribution of this reaction to
tyrosyl-mediated lipid peroxidation has never received attention to the
best of our knowledge. Moreover, the retention of the phenolic groups
in the tyrosine oxidation product, dityrosine, raises the possibility
of further oxidation. Herein we also report rate constants for
dityrosine oxidation by both MPO-I and MPO-II. These data will be
useful in evaluating the stability of the end product dityrosine, which
has been proposed as a possible marker for lipid peroxidation (30) or as an index of oxidative damage to proteins in vivo and in vitro(31, 32) . Since chloride is
considered the major substrate of myeloperoxidase in vivo, we
also investigated the steady state rates of tyrosine oxidation in the
presence and absence of chloride. By use of a sequential-mixing
stopped-flow instrument we have measured the rate constant for MPO-I
reaction with chloride previously unreported in the literature. These
results not only contribute quantitative support to the accumulating
evidence on the role of tyrosyl radicals in lipoprotein peroxidation
but also present differences in tyrosine oxidation by MPO compared with
other peroxidases.
Diluted hydrogen peroxide, obtained as
a 30% solution from BDH Chemicals, was standardized using the
horseradish peroxidase-catalyzed oxidation of iodide to
triiodide(38) . Concentrations were confirmed using absorbance
measurements at 240 nm where the extinction coefficient of
HO
is 39.4 M
cm
(39) . Ultrapure L-tyrosine was
obtained from Sigma. KCl (Aldrich) and the chemicals used for the
buffers (Fisher) were used without further purification. Aqueous
solutions were prepared using water purified through the Milli-Q system
(Millipore Corp.), and concentrations of solutes were determined by
weight.
Dityrosine was prepared using a combination of published
procedures (24, 40, 41) . 1.0 gram of L-tyrosine was dissolved in 910 ml of water, and then 50 ml of
0.1% HO
and 40 mg of horseradish peroxidase
(Type VI-A, Sigma) were added. The pH was adjusted to 9.2 using 6 M NaOH, and the mixture was incubated in a thermostated bath for 17
h at 37 °C. At the end of the reaction the pH was adjusted to 6.0
with concentrated HCl. The mixture was concentrated almost to dryness
in a rotatory evaporator under vacuum at 40 °C. Water was added to
the concentrate to a volume of 60 ml. The mixture was treated with 2 g
of Darco G-60 activated charcoal (Aldrich) and left standing overnight.
Centrifugation at 10,000 rpm for 15 min using the JA-20 rotor of
Beckman model J2-21 preparative centrifuge yielded a clear yellowish
brown supernatant. This was then applied to a precycled P-11 cellulose
phosphate cationic exchanger (Whatman) column (1.5
35 cm),
which had been previously equilibrated with 0.2 M acetic acid.
Elution of the column was performed with 0.2 M acetic acid
containing 0.5 M NaCl at a flow rate of 15 ml/h. Fractions of
3 ml were collected and evaluated spectrophotometrically at 280 and 310
nm. Samples of the major peak fractions were transferred on thin layer
silica gel plates (Whatman). After developing the plates in n-butyl alcohol/acetic acid/water (4:1:1, v/v) the fractions
were pooled that were positive for ninhydrin and with R
value (the ratio of the distance traveled by the band to the
distance traveled by the mobile phase) of about 0.15. The solution was
applied to a Dowex 50
8 (Terochem) column previously soaked in
0.1 M HCl. The column was extensively washed with water to
remove all NaCl and acetic acid. The dityrosine was subsequently eluted
with 2 M ammonia. Eluates were evaluated
spectrophotometrically as before. Samples of major peak fractions were
applied on thin layer silica gel plates, and the fractions with R
values of about 0.12 were pooled. The solvent
was removed in a rotatory evaporator under vacuum. The concentrate of
dityrosine was characterized by thin layer chromatography, ultraviolet
spectroscopy and fluorescence measurements(42) . Final
concentrations were determined by measuring absorbance at 315 nm using
an extinction coefficient of 5.2 mM
cm
at pH 7.5(43) .
The kinetics of the reactions of
MPO-I with tyrosine and dityrosine were carried out using the
sequential mixing mode of the stopped-flow apparatus. 1.0 µM of myeloperoxidase was premixed with 20 µM
HO
in 0.1 M phosphate buffer. After a
delay time of 20 ms the formed MPO-I was allowed to react with varying
concentrations of L-tyrosine or dityrosine, the final
concentrations of which were at least 10-fold in excess of the enzyme
intermediate. The time course of the reaction was followed by
monitoring the absorbance changes at 456 nm accompanying the formation
of MPO-II. This wavelength is also the isosbestic point between native
enzyme and MPO-I(36) .
The reaction of MPO-I with chloride
was investigated at 429 nm, the isosbestic point between MPO-I and
MPO-II, which also corresponds to the maximum change in absorbance as
MPO-I undergoes a two-electron reduction back to the native enzyme.
Pseudo-first order rate constants, k, were
determined using the single exponential curve-fit equation of the
Applied Photophysics software. Five or six determinations of rate
constants were performed for each reducing substrate concentration, and
the mean values were plotted against the substrate concentration. The
apparent second order rate constants for the MPO-I reactions were
calculated from the slopes using linear least squares regression
analysis (Enzfitter, Elsevier-Biosoft).
MPO-II reactions with L-tyrosine and dityrosine were carried out using the
single-mixing mode of the stopped-flow apparatus. Prior to the kinetic
experiments the stability of preformed MPO-II was studied by wavelength
scanning using the Beckman spectrophotometer. MPO-II was prepared by
adding a 50-fold excess of HO
to native enzyme
and then allowing this enzyme intermediate to react with tyrosine and
dityrosine under pseudo-first order conditions. Kinetic measurements
were made at 456 nm where the maximum change in absorbance is observed
as MPO-II is converted back to native enzyme. Pseudo-first order rate
constants were determined from seven or more exponential traces, and
second order rate constants were calculated as before.
Steady state
experiments on the oxidation of tyrosine by the
MPO/HO
system were conducted by following the
initial rate of formation of dityrosine. The reaction was monitored by
measuring the increase in fluorescence with time using wavelengths of
325 and 405 nm for excitation and emission. In a typical experiment one
syringe contained MPO and tyrosine in phosphate buffer while the other
contained H
O
. To determine the effect of
chloride, known concentrations of KCl solution were added to the
syringe containing H
O
. The final concentrations
after mixing were 25 nM MPO, 50 µM
H
O
, 0.2-1.0 mM tyrosine, and
0-0.125 M KCl. The initial rates were determined from
the slopes of the first linear portions of the traces obtained after
mixing all the components. The dead time of the instrument was
1.5
ms. Usually six to eight traces were recorded, and the mean values of
the initial rate of increase in fluorescence with time were calculated.
A calibration curve of fluorescence signal (volts) against dityrosine
concentration is linear up to 20 µM dityrosine. By
interpolation, initial rates in fluorescence units were converted to
rates in terms of dityrosine concentration. Double reciprocal plots as
well as Dixon plots were constructed to determine the type of
inhibition chloride exerts on tyrosine oxidation(44) .
Preliminary wavelength scans of myeloperoxidase in phosphate
buffer in the absence and presence of tyrosine or dityrosine revealed
no interference in the wavelengths to be used for transient kinetic
measurements (spectra not shown). Kinetic scans of the reaction between
native MPO and HO
in the presence of tyrosine
are shown in Fig. 1. The first scan is that of native enzyme,
and the second scan is that of MPO-I. The succeeding scans demonstrate
the rapid reduction of MPO-I to MPO-II. MPO-I formation takes place
within 10 ms after mixing, followed by its conversion to MPO-II, which
is completed in 100 ms. Full formation of MPO-I is achieved only in the
presence of a 20-fold excess of
H
O
(36) . However, since MPO-I is also
spontaneously reduced to MPO-II in the presence of excess
H
O
it was necessary to determine the rate of
reduction of MPO-I by tyrosine relative to its rate of spontaneous
decay. The instability of MPO-I necessitated the use of the sequential
mixing stopped-flow apparatus. The premixing of MPO and
H
O
led to MPO-I formation within 20 ms, and
before the MPO-I decayed it allowed the measurement of the rate of the
subsequent reaction of MPO-I with a reducing substrate.
Figure 1:
Rapid scan spectra
of MPO-I reduction to MPO-II by tyrosine. One syringe contained 1.0
µM MPO and 50 µM tyrosine in phosphate buffer
while the other had 20 µM HO
in
the same buffer. The arrows show the direction of absorbance
changes with time. The first, second, and last scans were taken 2.5,
10, and 100 ms after mixing.
The time
courses for the reduction of MPO-I to MPO-II may be followed by
monitoring the disappearance of MPO-I at 442 nm, the isosbestic point
between MPO and MPO-II, or the formation of MPO-II at 456 nm, the
isosbestic point between MPO and MPO-I. Rate constants obtained in both
cases are the same within experimental uncertainty(36) . We
chose to perform kinetic measurements for MPO-I reduction by tyrosine
at 456 nm because the absorbance change accompanying MPO-II formation
at this wavelength is larger and leads to smaller experimental errors.
A typical kinetic trace displaying single exponential character is
shown in the inset to Fig. 2. The apparent second order
rate constant was obtained from the slope of the plot of pseudo-first
order rate constants, k, against tyrosine
concentration (Fig. 2): (7.7 ± 0.1)
10
M
s
. A
value of (6.3 ± 0.2)
10
M
s
was obtained using
the single-mixing mode of the apparatus. However, the intercept of the
plot obtained using the single-mixing mode is bigger, 9.5 ± 1.8
s
, compared with 2.2 ± 1.2 s
(Fig. 2), obtained using the sequential-mixing mode. The
value of the intercept indicates the rate of the spontaneous reduction
of MPO-I by excess H
O
in the absence of
tyrosine. Thus, the use of sequential mixing stopped-flow minimized the
interference of MPO-I reduction to MPO-II by excess
H
O
.
Figure 2:
Pseudo-first order rate constants for
MPO-I reduction by tyrosine. The inset shows a typical trace
and curve fit (solid line) of the reaction followed at 456 nm
using sequential mixing mode. Final concentrations were 0.25 µM MPO, 5 µM HO
(µ =
0.1 M due to phosphate buffer, pH 7.4). The second order rate
constant was calculated from the slope. The standard deviations of the k
values ranged from 0.5 to
3.5%.
The rate constant for MPO-I reduction by tyrosine at pH 7.4 was higher than the rate at pH 4.5, the pH inside the phagocyte(45) . We performed pseudo-first order rate measurements at single tyrosine concentrations from pH 3.0 to 8.8, and the results are presented in Fig. 3.
Figure 3:
pH
dependence of pseudo-first order rate constants for MPO-I reduction by
tyrosine. Final concentrations were 0.5 µM MPO, 10
µM HO
, 50 µM tyrosine
in 0.1 M buffer. The buffers used were citrate (pH
3.0-5.4), phosphate (pH 6.0-7.8), and carbonate (pH
8.8).
While MPO-I is
thermodynamically competent to form tyrosyl radicals via its
one-electron oxidation of tyrosine(17) , the capability of
MPO-II to generate tyrosyl radicals was unknown(12) . MPO-II is
more stable than MPO-I(36, 46, 47) . A good
preparation of MPO-II is obtained by adding a 50-fold excess of
HO
to pure native enzyme. The MPO-II spectra
remained basically unchanged after 10 min (Fig. 4). Upon
addition of tyrosine, MPO-II is rapidly reduced to native enzyme. This
one-electron enzyme reduction must be accompanied by a one-electron
oxidation of the reducing substrate. These results prove that MPO-II is
also capable of oxidizing tyrosine and generating tyrosyl radicals.
Figure 4:
Wavelength scan of the reaction between
MPO-II and tyrosine. Scan A is for MPO (0.5 µM in
0.1 M phosphate buffer). Consecutive scans B (MPO-II)
were taken within a time period of 10 min after adding 25
µM HO
to MPO. Scan C was
taken immediately after the addition of 50 µM tyrosine to
MPO-II.
Because of the relative stability of MPO-II we were able to conduct
rate measurements of the reaction of MPO-II with tyrosine using the
single-mixing mode of the stopped-flow apparatus. The monophasic
exponential decrease in absorbance at 456 nm with time as MPO-II is
converted back to native enzyme yielded k values
that were plotted against tyrosine concentration as before (data not
shown). The plot was linear up to 250 µM tyrosine, and the
intercept is zero within the standard deviations of the measurements.
The second order rate constant for the reaction of MPO-II with tyrosine
obtained from the slope of the plot was (1.57 ± 0.06)
10
M
s
.
Dityrosine has been detected in the MPO/HO
system of human neutrophils and macrophages as a product of
tyrosyl radical coupling (30) . Aside from the dimer, other
polymeric products have been identified in sytems employing horseradish
peroxidase (25) and ovoperoxidase(48) . It was of
interest to determine whether MPO would further catalyze the oxidation
of dityrosine since the phenolic groups are retained in the dimer.
Dityrosine was prepared enzymatically, purified, and characterized (42) . For the kinetic measurements it is important to work
with authentic dityrosine freed of tyrosine impurities. Dityrosine is
easily monitored by its characteristic fluorescence at 400
nm(49) . It also shows maximum absorbance at 285 nm in acid
solution and at 315 in alkaline solution(26) . Dityrosine
yields a single ninhydrin-positive spot on silica gel with R of 0.12 when using n-butyl
alcohol/acetic acid/water (4:1:1 v/v) as a solvent
system(42, 50) . All of these criteria were satisfied
by the dityrosine used in this work.
The addition of dityrosine to
preformed MPO-II resulted in conversion of MPO-II to native enzyme,
although not as fast compared with the reaction of tyrosine. A shoulder
at 456 nm in the wavelength scans (Fig. 5, inset)
indicates that MPO-II reduction is not complete within the time period
of the scan. Similar rate measurements under pseudo-first order
conditions were conducted for the reactions of MPO-I and MPO-II with
dityrosine. The second order rate constant determined from the slope of
the plot of k against dityrosine concentration
for the MPO-II reaction is (7.5 ± 0.3)
10
M
s
(Fig. 5). The
second order rate constant for the MPO-I reaction with dityrosine was
more than 2 orders of magnitude larger: (1.12 ± 0.01)
10
M
s
.
Figure 5:
Determination of rate constant for the
reaction of MPO-II with dityrosine. Pseudo-first order rate constants
were determined upon mixing MPO-II (prepared by adding 25
µM HO
to 0.5 µM MPO)
with varying concentrations of dityrosine. The inset shows
wavelength scans for the reaction. Scan A is for 0.5
µM MPO in 0.1 M phosphate buffer; scan B (MPO-II) was taken after 10 µM H
O
was added to the native enzyme; and scan C was taken
after adding 50 µM dityrosine to
MPO-II.
The major reducing substrate for MPO is considered to be chloride,
the oxidation product of which is hypochlorous acid(8) . It has
been suggested that chloride inhibits the initial rate of oxidation of
tyrosine by myeloperoxidase(30) . It is relevant to evaluate
the relative rates of tyrosine and chloride oxidation by MPO-I. By
using sequential mixing stopped-flow measurements we determined the
second order rate constant for MPO-I oxidation of chloride to be (4.7
± 0.1) 10
M
s
. Unlike previous plots, which had nearly zero
intercepts ( Fig. 2and Fig. 5, for example), the plot of k
versus chloride concentration yielded
an intercept of (31 ± 4) s
(data not shown).
We were also interested in the type of inhibition chloride exerts on
tyrosine oxidation. This was investigated under steady state conditions
by monitoring the rate of formation of the dityrosine oxidation product
in a system containing MPO, HO
, tyrosine, and
chloride. The rate of dityrosine formation may be followed by
monitoring absorbance changes at 315 nm (43) or fluorescence
changes using 325 and 405 nm as excitation and emission wavelengths.
The fluorescence signal is larger, so we chose it over absorbance to
measure initial rates of dityrosine formation. Fig. 6shows that
tyrosine does not contribute to the fluorescence reading. The
concentration of dityrosine in the reaction mixture was interpolated
from a calibration curve of purified dityrosine (Fig. 6, inset).
Figure 6: Fluorescence spectra of dityrosine. The excitation wavelength was set at 325 nm. Tyrosine does not fluoresce when exposed to 325-nm light. The inset shows the calibration curve used for calculating dityrosine concentration from fluorescence readings.
The initial rate of tyrosine oxidation was measured
as a function of tyrosine concentration, and the results are shown in Fig. 7. Obviously, the plot is not hyperbolic and therefore does
not yield corresponding Michaelis-Menten parameters. Double reciprocal
plots of initial rates against tyrosine concentration increase in slope
as the concentration of chloride increases (Fig. 8). A secondary
plot of the slopes from the double reciprocal plots against chloride
concentration (Fig. 8, inset) yields an inhibitor
constant K equal to 0.16 M. Values of K
are usually obtained accurately by using Dixon
plots(51) , i.e. plots of the reciprocal of initial
rate against inhibitor concentration at various fixed concentrations of
substrate. The family of Dixon plots in Fig. 9suggests either
competitive or linear mixed-type inhibition(44) , and from the
point of intersection we obtained the inhibition constant of chloride, K
= 0.18 ± 0.02 M; and the
maximum velocity for tyrosine oxidation, V
= 1.7 ± 0.1 µM dityrosine
s
.
Figure 7:
Steady state initial rates of dityrosine
formation. Final concentrations were 25 nM MPO, 0.1
mM HO
at 0.1 M phosphate. The
initial rates were determined from the slope of the initial portion of
the trace of increase in fluorescence with time (inset, 1
mM tyrosine was used).
Figure 8:
Double reciprocal plots for chloride
inhibition of dityrosine formation. Final concentrations were 25 nM MPO, 50 µM HO
at 0.1 M phosphate; chloride concentrations (in M) were 0
(
), 0.06 (
), and 0.125 (
). Inset is a
replot of the slopes from the reciprocal plots against chloride
concentration. The x intercept gives the negative value of K
.
Figure 9:
Dixon plots for chloride inhibition. Final
concentrations are as in Fig. 8. Tyrosine concentrations (in
mM) were 0.4 (), 0.6 (
), 0.8 (
), and 1.0
(
).
The peroxidase-catalyzed oxidation of tyrosine has recently attracted renewed interest due to its involvement in lipid and protein oxidation(12, 27, 31) . The oxidation of tyrosine has also been implicated in the biosynthesis of thyroxine and melanin (43) and in the cross-linking of structural proteins via the dityrosine linkage (for a review see (42) ). Previous investigations of the kinetics of tyrosine oxidation have been performed using lactoperoxidase(43, 52) , horseradish peroxidase(53, 54) , and thyroid peroxidase(52, 55) . Our present work provides the first report of the rate constants for the oxidation of tyrosine as catalyzed by myeloperoxidase. These results are significant in lieu of the findings that MPO has been identified as a physiological catalyst for lipid peroxidation in low density lipoproteins via tyrosyl radicals(12, 23) .
While all hemoprotein
peroxidases are generally considered to catalyze oxidation reactions in
a similar manner, Table 1shows varying values in the rate
constants for the oxidation of tyrosine by several peroxidases under
similar pH and temperature conditions. This reflects differences in the
effectiveness of various peroxidases in catalyzing the oxidation of
tyrosine. Horseradish peroxidase is the least effective catalyst for
tyrosine oxidation. Among the three mammalian peroxidases, thyroid
peroxidase has the lowest rate constant for compound I reduction by
tyrosine. This is to be expected because the primary reaction catalyzed
by thyroid peroxidase is iodination of the tyrosyl residues in
thyroglobulin; therefore, tyrosine oxidation may not be as important.
Moreover, thyroid peroxidase has a unique property of catalyzing the
two-electron oxidation of tyrosine(55) , which differentiates
it from lactoperoxidase and myeloperoxidase, which oxidize tyrosine via
two successive one-electron processes. The rapid spectral scans of the
MPO/HO
system in the presence of tyrosine (Fig. 1) clearly show that MPO-I undergoes a one-electron
reduction to MPO-II followed by the slower one-electron reduction of
MPO-II to native enzyme (Fig. 4).
The rate constant for lactoperoxidase compound-I reaction with tyrosine could only be estimated by previous workers (52) due to the inherent instability of its compound I. We were able to overcome this problem in our transient kinetic measurements through the use of a sequential mixing stopped-flow apparatus. Our data establish that myeloperoxidase is the most effective among the mammalian peroxidases in catalyzing both the one-electron reductions of its compounds I and II by tyrosine. Our results also show that tyrosine could contribute to the turnover of MPO-II to native enzyme in the absence of other reducing substrates.
Another striking difference displayed by these peroxidases in their oxidation of tyrosine is the pH at which the maximum rate is observed for compound I reduction. For horseradish peroxidase, the maximum rate is at pH 9.6(53) , while for lactoperoxidase it is at pH 8.2(43) . Fig. 3indicates that for MPO, the maximum rate is achieved at the physiological pH of 7.4. In a related study of the pH dependence, the formation of dityrosine, the stable primary product of the myeloperoxidase-catalyzed oxidation of tyrosine, was found to be highest at pH 7.5-8.0(30) . Apparently, oxidation of tyrosine requires deprotonation of its phenolic hydroxyl group(56) . Thus, inside the acidic environment of the phagosome (45) the predominant reaction is not likely tyrosine oxidation but rather chloride oxidation by MPO-I to form HOCl. Moreover, it has been demonstrated that only at acidic pH is there direct contact between chloride and ferryl oxygen in MPO-I resulting in a maximum rate of HOCl formation(57) .
MPO-I and MPO-II are each able to oxidize tyrosine in a one-electron reaction to give tyrosyl radicals. Long-lived tyrosyl radicals have also been produced by chemical oxidation of tyrosine using ferricyanide and characterized by electron spin resonance(58) . However, the readily detectable product of tyrosine oxidation is o,o`-dityrosine formed by coupling of phenoxy radicals(25) . Dityrosine is intensely fluorescent (Fig. 6), and in proteins it has been found to be resistant to proteolysis(31) , to borohydride treatment(26) , and to acid hydrolysis(30) . Because of its apparent stability dityrosine has been proposed as a chemical marker of cumulative exposure of proteins to metal-catalyzed oxidation in vitro and in vivo(32) , an index of organismal oxidative stress(31) , and an indication of targets where phagocytes inflict oxidative damage in vivo(30) . However, it is also known that extensive action of peroxidases on tyrosine results in the formation of other oxidation products such as trityrosine and other polymers, some of which are also fluorescent(25, 30, 43, 48) . This is probably the reason why the steady state rates of dityrosine formation measured by the increase in fluorescence did not reach saturation but continued to increase with time (Fig. 7).
Since the phenoxy groups are retained in dityrosine it is likely
that dityrosine can be further oxidized by the MPO/HO
system. The inset to Fig. 5shows that dityrosine
can reduce MPO-II to native enzyme, although the rate would not seem
physiologically relevant. On the other hand the rate constant for MPO-I
reaction with dityrosine yields a value, (1.12 ± 0.01)
10
M
s
,
which is not significantly lower than the reaction of MPO-I with
tyrosine. The lower rate constant for the more bulky dityrosine
substrate is consistent with the finding that aromatic substrate
molecules bind at the distal heme pocket of myeloperoxidase (59) and that this site exhibits considerable steric
hindrance(17) . The fast oxidation of dityrosine by MPO-I
suggests that dityrosine may not be a very stable end product and that
it should be used with caution as a marker for oxidative damage,
particularly in the presence of the MPO/H
O
system.
Since the major physiological substrate of MPO is
considered to be chloride, we also evaluated the relative rates of
tyrosine and chloride oxidation by MPO-I. There have been difficulties
in obtaining the rate constant for the two-electron oxidation of
chloride by MPO-I. Full formation of MPO-I is achieved only in the
presence of at least a 20-fold excess of HO
over MPO(30) . But with excess
H
O
, MPO-I is reduced at a very fast rate to
MPO-II, making it difficult to measure the rate constant for the MPO-I
reaction with chloride. We were able to overcome this problem by using
the sequential mixing mode of the stopped-flow apparatus. The second
order rate constant for chloride peroxidation by MPO-I, (4.7 ±
0.1)
10
M
s
, is about an order of magnitude higher than the
rate constant for tyrosine oxidation by MPO-I and about the same value
as the rate constant for the formation of an MPO-chlorinating
intermediate (2.8 ± 1.2)
10
M
s
(60) .
However, unlike the other plots of k
against
substrate concentration, which had zero intercepts within experimental
error ( Fig. 2and Fig. 5, for example), the plot for the
chloride reaction with MPO-I yielded a considerable y intercept (31 ± 4) s
. This suggests a
reversible reaction. In fact, the oxidation product of chloride, HOCl,
can undergo a rapid reaction with native MPO to give MPO-I, which is
ostensibly the reverse of the chloride peroxidation
reaction(17, 18) .
For the chloride inhibition
studies we constructed both double reciprocal and Dixon plots, which
are used to identify the type of inhibition and to determine the
inhibition constant, K(51) . The two types
of plots only establish that the inhibition is neither noncompetitive
nor uncompetitive. The data are characteristic of either purely
competitive or mixed-type inhibition(44) . The replot of the
slopes of the Dixon plots against reciprocal of substrate (tyrosine)
concentration did not umambiguously predict which type of inhibition is
occurring. The Cornish-Bowden plot (61) (graphs not shown) did
not clarify the kind of inhibition either. Indeed it may be difficult
to characterize the type of inhibition in such a complex system where
the enzyme is in contact with three substrates:
H
O
, tyrosine, and chloride. A simple
interpretation of plots used for inhibition studies is not possible for
multisubstrate systems(61) , and especially where the products
(HOCl and dityrosine) are also substrates. H
O
and chloride have been shown to be competitive inhibitors of each
other(62, 63, 64) . The substrate inhibition
patterns of these studies suggest the existence of two binding sites
for chloride; one is a substrate binding site and the other an
inhibitor binding site. The binding of chloride to its substrate
binding site results in the production of HOCl, whereas its binding to
the inhibitor binding site leads to competitive inhibition of
H
O
binding to native MPO. Inhibition by
chloride almost certainly involves coordination to the heme because
MPO-I has a ferryl oxygen occupying the sixth coordination position and
possesses no available chloride binding sites. The catalytic site for
chloride peroxidation is most likely located within the distal heme
cavity (57) or at the heme periphery (17, 65) . Recently, EPR spectroscopy and model
building revealed that the hydrophobic character of the entrance to the
distal cavity of MPO allows favorable interaction with aromatic
molecules (59) such as tyrosine. If indeed the chloride
substrate binding site is in the distal heme pocket, then it would
compete with tyrosine. However, the large value of K
we obtained in our steady state studies suggests that binding of
chloride is very weak, presumably because of the hydrophobic nature of
the pocket. That would explain why tyrosine oxidation takes place at
considerable rates even in the presence of high concentrations of
chloride. These results are consistent with the findings that tyrosine
oxidation occurs even in the presence of physiological concentrations
of chloride (12, 30) and that LDL oxidation by tyrosyl
radicals is largely unaffected by as high as 0.1 M chloride
present in the plasma(23) .
In conclusion, both MPO-I and MPO-II are able to react with tyrosine via one-electron oxidations and therefore generate tyrosyl radicals. The relatively fast rate of the MPO-I reaction with tyrosine substantiates the conjecture that tyrosine oxidation by MPO-I takes place in vivo and may be physiologically relevant. Tyrosine also competes very effectively with the major myeloperoxidase substrate chloride. That tyrosine can also reduce MPO-II to native enzyme ensures turnover of the enzyme even in the absence of other reducing substrates. The tyrosyl radical formed in both reactions is a diffusible catalyst that can convey oxidizing potential away from the active site of the heme enzyme. A protein tyrosyl radical has been implicated in cross-linking of proteins(12) , in initiating lipid peroxidation in LDL (23) and modifying HDL to protect the arterial wall against cholesterol accumulation(27) . The readily detectable product of tyrosine oxidation is dityrosine formed by the coupling of tyrosyl radicals. Our study revealed that since dityrosine is further oxidized at a very fast rate by MPO-I caution must be exercised when using it as a quantitative index of lipoprotein modification. It would appear to measure a lower limit of lipoprotein modification.