(Received for publication, July 30, 1996, and in revised form, October 29, 1996)
From the Nitrogen Fixation Laboratory, John Innes
Centre, NR4 7UH Norwich, United Kingdom and the ¶ Departamento
de Biología Vegetal (Fisiología Vegetal) and
** Departamento de Bioquímica y Biología Molecular
A, Universidad de Murcia, 30100 Murcia, Spain
The kinetics of the catalytic cycle and
irreversible inactivation of horseradish peroxidase C (HRP-C) reacting
with m-chloroperoxybenzoic acid (mCPBA) have been studied
by conventional and stopped-flow spectrophotometry. mCPBA oxidized
HRP-C to compound I with a second order-rate constant
k1 = 3.6 × 107
M1 s
1 at pH 7.0, 25 °C.
Excess mCPBA subsequently acted as a one-electron reducing substrate,
converting compound I to compound II and compound II to resting, ferric
enzyme. In both of these reactions, spectrally distinct, transient
forms of the enzyme were observed (
max = 411 nm,
= 45 mM
1 cm
1 for compound I with
mCPBA, and
max = 408 nm,
= 77 mM
1 cm
1 for compound II with
mCPBA). The compound I-mCPBA intermediate (shown by near infrared
spectroscopy to be identical to P965) decayed either to compound II in
a catalytic cycle (k3 = 6.4 × 10
3 s
1) or, in a competing inactivation
reaction, to verdohemoprotein (ki = 3.3 × 10
3 s
1). Thus, a partition ratio of
r = 2 is obtained for the inactivation of ferric HRP-C
by mCPBA. The intermediate formed from compound II with mCPBA is not
part of the inactivation pathway and only decays via the catalytic
cycle to give resting, ferric enzyme (k5 = 1.0 × 10
3 s
1). The data are compared
with those from earlier steady-state kinetic studies and demonstrate
the importance of single turnover experiments. The results are
discussed in terms of the physiologically relevant reactions of plant
peroxidases with hydrogen peroxide.
Horseradish peroxidase (HRP)1 (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) is an extracellular plant enzyme involved in the formation of free radical intermediates for the polymerization and cross-linking of cell wall components, for the oxidation of secondary metabolites essential for certain pathogenic defense reactions, and for the regulation of cell growth and differentiation (1). There are more than 30 isoforms of HRP, which are usually classified, according to isoelectric point, into three major groups: acidic, neutral, and basic (2). The slightly basic (cationic) horseradish peroxidase C (HRP-C) is the most studied isoform, since it constitutes approximately 50% of the peroxidase content of horseradish root and is commercially available because of its use in clinical analysis and biotransformations (see Refs. 3-5 for reviews of its structure and function).
HRP-C is a monomeric (Mr of 33,922),
glycosylated (18% by mass) enzyme that contains a single high spin
ferric protoporphyrin IX prosthetic group and two calcium ions (6-8)
that are necessary for competent folding of the recombinant enzyme
after expression in Escherichia coli (9). The catalytic
cycle is initiated by a rapid (k1 > 107 M1 s
1)
2e
oxidation of the enzyme by hydrogen peroxide (or other
organic peroxides) to give a green enzyme intermediate, compound I,
with the heme iron oxidized to the oxyferryl state (Fe(IV)=O) and a
-cation radical on the porphyrin ring. Compound I formation involves at least two reactions; formation of an intermediate
enzyme-hydroperoxide complex (10), followed by heterolytic cleavage of
the oxygen-oxygen bond. Histidine 42 and arginine 38, located in the
distal heme cavity, are key residues that modulate both these reactions
(11, 12). In order to complete a peroxidation cycle, compound I is reduced back to the resting Fe(III) state by two successive single electron transfer reactions from reducing substrate molecules, the
first yielding a second enzyme intermediate, compound II. Both of these
reactions can yield free radical products that can undergo subsequent
chemistry. The rate of the peroxidation cycle usually depends on the
nature of the reducing substrate, with the reduction of compound II to
resting enzyme being rate-limiting (3).
In the absence of reducing substrates, excess hydrogen peroxide can
react with compound I as an electron donor (reductant) in a
catalase-like two-electron process that results in the formation of
molecular oxygen or in two single-electron transfers in which compound
II, compound III (Fe(II)-O2), and superoxide radical anion
(O2) are formed (13-15). Importantly in the context of this paper, a competing enzyme inactivation takes place that results in the
generation of a number of inactive chromophores with the final product
being a verdohemoprotein, P670 (16-19). A global reaction scheme that
includes all the previously identified partial reactions of the
catalytic cycle and the inactivation pathway has been developed using
experimentally determined steady-state kinetic parameters for the
HRP-C/H2O2 system (20). It was concluded that
the catalase-like and compound III generating activities of HRP-C
protect against inactivation and that the extent of inactivation is
determined by the reactivity of a common intermediate, the complex
formed between compound I and a second equivalent of
H2O2 (18, 20). Consequently, with excess
H2O2 as the only reducing substrate, the enzyme
is completely inactivated after only ~600 turnovers (20). A
simplified form of the reaction scheme has recently been developed to
enable a more rapid, comparative analysis of data for isoenzymes from
diverse sources (21) and to determine the effects of selected mutations
in recombinant HRP-C (22).
A complementary approach to understanding the inactivation mechanism of
HRP-C utilizes the competent, xenobiotic substrate, meta-chloroperoxybenzoic acid (mCPBA) instead of hydrogen
peroxide. Compound I formation results in the release of the parent
carboxylic acid instead of water (23) (Scheme I). Importantly, HRP-C in the ferric, compound I, and compound II states has a high affinity for
mCPBA possibly because of its structural resemblance to reducing organic substrates such as benzhydroxamic acid (24-26) and
indole-3-acetic acid (27, 28). These affinities permit single turnover
experiments, with an excess of enzyme over substrate, using rapid-scan
stopped-flow spectrophotometry. These conditions allow a precise
determination of the values of the rate constants for the partial
reactions shown in Table I in the absence of subsequent reactions that would occur at significant rates with an excess of mCPBA. In addition, the absence of the catalase-like cycle (13, 26, 29) causes the average
number of turnovers effected by each molecule of enzyme before
inactivation (r) to drop to very low values, typically 2 or
3 (22, 26, 30). This simplifies the kinetic analysis and aids the
identification of intermediates in both the catalytic cycle and on the
inactivation pathway, facilitates simulations of the complex absorbance
changes, and allows a more rigorous test of our proposed mechanism of
inactivation shown in Scheme I than was possible using
pseudo-steady-state kinetics. In addition it has allowed us to show
unambiguously that the reactivity of the complex formed between
compound I and mCPBA is the key intermediate that determines the extent
of inactivation. The results with mCPBA validate our earlier
conclusions for the reactions of HRP-C with hydrogen peroxide, where it
was only possible to use pseudo-steady-state kinetics, and allow us to
discuss with greater confidence the physiological implications.
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Horseradish peroxidase isoenzyme C was purchased
from Biozyme (Type HRP-4B) and used without further purification.
Characterization by electrophoresis and isoelectric focusing (21)
revealed a band for peroxidase activity at pI 8.5, typical of a basic
isoenzyme. Its concentration was determined spectrophotometrically
using 403 nm = 102 mM
1
cm
1. A specific activity of 1,322 µmol
mg
1 min
1 was determined using 0.2 mM H2O2 and 0.5 mM ABTS
as the reducing substrate in 50 mM glycine-HCl buffer, pH
4.5 at 25 °C. The preparations used for this study had ratios of
absorbance at 403/280 nm (RZ value) of 3.2. mCPBA in the solid state
(Aldrich) containing m-chlorobenzoic acid as an impurity was
recrystallized from light petroleum ether (boiling point
40-60 °C)/diethylether (3:1, v/v) as described by Davies et
al. (24), to give material that was more than 96% pure (NMR
analysis and single melting point at 92-94 °C). Solutions of mCPBA
were made up in 1:1 (v/v) absolute ethanol/water immediately prior to
use. The concentration was determined using
232 nm = 8,940 M
1 cm
1 allowing for the
percentage purity obtained by NMR (26). Reagent grade
H2O2 (30% v/v) was obtained from BDH, and its
concentration determined spectrophotometrically using an
240 nm of 43.6 M
1
cm
1 (31). ABTS was supplied by Sigma as a diammonium
salt, and solution concentrations were determined using a
340 nm of 36 mM
1
cm
1 (32).
Transient kinetics were monitored with a stopped-flow spectrophotometer (model SF-51), Hi-Tech Scientific, Salisbury, United Kingdom. Data were recorded through an RS232 interface with a microcomputer. Stopped-flow rapid-scan spectrophotometry was carried out with the same sample handling unit equipped with an MG-6000 diode array system. Spectrophotometric measurements were made with a Perkin-Elmer Lambda-2 UV-visible spectrophotometer controlled by an Olivetti PCS/386SX computer running dedicated software. The temperature was controlled at 25 or 10 °C using a Techne C-400 circulating bath with an integral heater-cooler system.
Pre-steady-state KineticsCompound I formation was monitored at 395 nm (isosbestic for compounds I and II). Compound II formation from compound I was monitored at 412 nm (isosbestic for compound II and the resting enzyme). Compound I was generated by mixing enzyme with an equimolecular amount of H2O2 in a simple pre-mixing device attached to one of the transport syringe ports on the stopped-flow apparatus and transferred directly to a drive syringe for use within 5 min of preparation. Compound II was made from compound I (prepared as described above) by oxidative titration with ascorbic acid. The disappearance of compound II was monitored at 424 nm (the isosbestic for compound I and the ferric enzyme). The formation of verdohemoprotein was followed at 670 nm. The determination of the reaction rates were carried out under single turnover conditions with [E]0 > [S]0 (33). Pre-steady-state kinetic experiments were performed in 10 mM sodium phosphate buffer, pH 7.0, and the data analyzed by fitting the absorbance-time curves to exponential functions using a least-squares minimization procedure.
SpectrophotometryThe detection of enzyme intermediates and final products from reactions of ferric enzyme, compound I or compound II were carried out by mixing these species with a stoichiometric amount of mCPBA. Transient and final spectra were deconvoluted using a SPECFIT Global Analysis computer program (Spectrum Software Associates, Chapel Hill, NC). All experiments were in 10 mM sodium phosphate buffer, pH 7.0.
Kinetic SimulationsThe kinetics of the reaction mechanism described in Scheme I is defined by a set of differential equations, whose numerical integration was carried out using a KSIM computer program provided by Dr. N. Millar. The differential equations are shown in Equations 1-6 below.
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(Eq. 1) |
![]() |
(Eq. 2) |
![]() |
(Eq. 3) |
![]() |
(Eq. 4) |
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
Experimentally determined values of the equilibrium and rate constants were assigned to the partial reactions defined in Scheme I. The kinetics of the intermediates and products defined in Schemes II, III, and IV (shown in Table I) were simulated using the corresponding differential equations.
Formation of verdohemoprotein
(P670), with its characteristic absorption maximum at 670 nm, is
diagnostic of peroxidase inactivation (19). The titration data shown in
Fig. 1 with both spectrophotometric and enzyme activity
monitoring show that at least two equivalents of mCPBA were required
for its formation starting from HRP-C in the ferric state (Fig. 1). The
loss of enzymatic activity is clearly proportional to the extent of
P670 formation. These findings are consistent with the first equivalent
mCPBA being used to form compound I and the second to inactivate the
enzyme and suggest that compound I is a common intermediate in the
catalytic cycle and at the beginning of the inactivation pathway
(Scheme III shown in Table I). It can also be calculated from Fig. 1
that 8 mol of mCPBA are needed to completely inactivate 1 mol of enzyme
([substrate]/[enzyme] ratio = 70 µM/8.7
µM = 8.0). These equivalents are distributed between the
catalytic cycle and the inactivation pathway (Scheme I).
Compound I Formation
The reaction of HRP-C with a
stoichiometric concentration of mCPBA yielded compound I with a
UV-visible spectrum that was identical to that described for the
reaction of this enzyme with hydrogen peroxide (23). No differences in
the stability of this compound I were observed when compared with the
compound I formed with hydrogen peroxide under the same conditions. The
kinetics of the reaction of HRP-C with mCPBA to form compound I have
been shown by Dunford and Hewson (34) to have a viscosity dependence characteristic of a diffusion limited reaction
(k1 = 8.2 × 107
M1 s
1 independent of pH in the
range 5.0-6.8). Under single turnover conditions
([HRP-C]0 > [mCPBA]0) (Fig.
2), we have determined that k1 = (3.6 ± 0.6) × 107 M
1
s
1 at pH 7.0 (Table I). Under these
conditions, no inactivation of the enzyme occurred nor was any
verdohemoprotein formed.
The Reaction of mCPBA with Compound I
The two competing
reactions that the complex formed between compound I and mCPBA
undergoes (Scheme III shown in Table I) are proposed to explain the
relationship between catalytic cycle and the inactivation pathways. In
order to detect this transient intermediate and to determine the
kinetics and mechanism of its formation and decay, the reaction of
compound I with mCPBA was studied under stoichiometric conditions. Fig.
3A shows the formation of compound I-mCPBA
intermediate and its subsequent decay to compound II and P670. The
spectrum of the compound I-mCPBA complex was recorded after a 1-s
reaction time and showed maxima at 411, 531, 556, and 649 nm and a
shoulder at 615 nm (Fig. 3B). The extinction coefficient in
the Soret region was calculated to be 45 mM1
cm
1 using a global analysis with the computer program
SPECFIT. A similar spectrum to that assigned to the compound I-mCPBA
complex (Fig. 3B) was obtained in the reaction of compound I
with m-chlorobenzoic acid, the parent carboxylic acid
(not-peroxo form) of mCPBA (Fig. 4). This reaction when
followed at 405 nm showed monophasic kinetics with a second-order rate
constant of 5.3 × 103 M
1
s
1 and a dissociation constant of 0.25 mM.
These results indicate that the complex formed between compound I and
mCPBA is a real enzyme-substrate complex and could serve as a model for
the HRP-C compound I-reducing substrate complex, which cannot be
detected with conventional reducing substrates such as guaiacol or
ABTS.
The enzyme species designated P940 and P670 have been observed
previously in the reaction of HRP-C with an excess of hydrogen peroxide
(19) and other peroxides (16-18). Another species, P965, has also been
observed in the reaction of HRP-C with m-nitroperoxybenzoic acid (18). These authors proposed a sequential mechanism in which P965,
formed directly from the reaction of compound I with m-nitroperoxybenzoic acid, subsequently reacted irreversibly
with a second equivalent of the peroxide to give initially form P940 and then finally P670. In order to clarify the number of enzyme intermediates in the inactivation pathway, we have studied the reaction
of compound I with mCPBA in the near infrared region (Fig.
5). We have been restricted to using conventional
spectrophotometry with the first observation after manual mixing at
~15 s since our stopped-flow apparatus has a long wavelength limit of
700 nm. During the course of the reaction, an isosbestic point is maintained at 735 nm. The kinetics of the decrease in absorbance at 965 nm and the increase at 670 nm were identical and were fitted to a
single exponential function with kobs = 4 × 103 s
1 at 10 °C, independent of the
concentration of mCPBA (Fig. 5, inset). At the beginning, an
increase in absorbance at 965 nm took place as consequence of the
compound I-mCPBA complex formation. These data strongly suggest that
the compound I-mCPBA complex is P965, which is the only intermediate
that is detected when compound I is converted to verdohemoprotein
(P670) by reaction with one equivalent of mCPBA. The similarity in the
spectra of the complexes formed between compound I and both mCPBA and
m-chlorobenzoic acid (Figs. 3B and 4,
respectively) points to a chemical nature of P965 related to an
unreacted enzyme-substrate intermediate whose formation is reversible,
as was previously suggested for the (compound
I-m-nitroperoxybenzoic acid) complex (18). The subsequent
chemical structural change probably take place later, during the
transition P965
P670.
However, not all of the P965 is converted to P670 due to the competing
decay reaction that yields compound II (Scheme III shown in Table I).
Under single turnover conditions, with the first step equilibrating
rapidly (k2[mCPBA] + k2
k3, ki), the relaxation times are given by Equations 7 and 8.
![]() |
(Eq. 7) |
![]() |
(Eq. 8) |
|
In order to determine the individual values of
k3 and ki, we have measured
the relative amounts of compound II and P670 formed under single
turnover conditions as a function of increasing concentration of mCPBA
(Fig. 8). The final concentration P670 was determined
directly from the increase in absorbance at 670 nm using a calculated
670 nm = (19.6 ± 2.0)
mM
1 cm
1. The final
concentration of compound II could not be measured spectrophotometrically since several forms of the enzyme absorb in the
Soret region. Instead, it was estimated from the remaining enzymatic
activity, using the ABTS oxidation assay. The data in Fig. 8 show that
both determinations give a good linear correlation, indicating that the
distribution of compound I-mCPBA into compound II (67 ± 3)% and
P670 (33 ± 2)% was independent of the initial concentration of
the complex. This distribution can be defined as the partition ratio
(r = k3/ki = 2.0) and allows the values of k3 and
ki to be calculated in good agreement with the
values obtained previously under pseudo-steady-state conditions (Table
II) (26). In addition, our titration data (Fig. 1) show that 8 mol of
mCPBA were necessary to completely inactivate 1 mol of enzyme.
According to the calculated partition ratio of 2, the reaction
mechanism depicted in Scheme I is consistent with this stoichiometry.
Additional consumption of mCPBA in the inactivation pathway would
result in a different titration value. In consequence, only one
molecule of mCPBA is required to react with compound I for some
inactivation to be observed, and this is not consistent with other
mechanisms suggesting that two molecules of hydroperoxide from compound
I are needed in order to convert the enzyme to inactive P670 state
(18).
The Reaction of mCPBA with Compound II
An inactivation
pathway originating with compound II has been proposed previously (19).
In order to further investigate this proposal, we have studied the
reaction of compound II with stoichiometric and excess concentrations
of mCPBA using conventional diode-array and stopped-flow
spectrophotometry. Compound II with characteristic absorption maxima at
417, 529, and 556 nm was generated from compound I by the addition of
one equivalent of ascorbic acid. When compound II reacted with one
equivalent of mCPBA, a transient spectrum of an intermediate (assumed
to be the compound II-mCPBA complex) was initially observed. This
spectrum subsequently converted to that characteristic of the native
ferric state with an isosbestic point maintained at 411 nm (Fig.
9). The failure to detect any P670 product strongly
suggests that only compound I and not compound II is involved in the
initiation of the inactivation reaction. Under these conditions and
with a 10-fold excess of mCPBA, no spectroscopic evidence for transient
compound III formation was obtained. Thus the ferric enzyme product is
probably formed by single electron transfer from mCPBA to compound
II.
The kinetics of the reaction of compound II with mCPBA were studied
under single turnover conditions with increasing concentrations of
compound II at a fixed substoichiometric concentration of mCPBA. The
time dependence of the absorbance change at 403 nm showed an initial
decrease with a minimum at 25 s that corresponds to the formation
of the transient intermediate, compound II-mCPBA. This was followed by
an increase in the absorbance due to the formation of native ferric
enzyme. Assuming compound II-mCPBA complex formation occurs as a rapid
pre-equilibrium prior to electron transfer,
(k4[mCPBA] + k4
k5), the observed first order rate constants for
the two reactions,
1 and
2, are given
by Equations 9 and 10 (35).
![]() |
(Eq. 9) |
![]() |
(Eq. 10) |
Fig. 10 shows the linear dependence of
1 on the concentration of compound II under
pseudo-first-order conditions ([compound II]0
[mCPBA]0). The slope and intercept give the values of
k4, k
4, and
K4 (Table II). The electron transfer from mCPBA to compound II within the complex was very slow (
2 = 1.0 × 10
3 s
1), as shown by the
kinetics of the appearance of native ferric enzyme with monitoring at
403 nm (data not shown). Under these conditions, the low value of
K4 = 5.7 × 10
7 M
results in
2 being independent of compound II
concentration and equal to k5. This value of
k5 is in agreement with that obtained under
steady-state conditions with ([mCPBA]0
[HRP-C]0) (Table II) (26).
The experiments described above have shown conclusively that mCPBA induced inactivation of HRP-C occurs by the mechanism described in Scheme I. The data are not consistent with the mechanism proposed by Marklund (17) nor with the modified mechanism of Nakajima and Yamazaki (18). Our kinetic and titration data clearly demonstrate that only two molecules of mCPBA are necessary to form inactive verdohemoprotein from the native ferric state of HRP-C. In addition, other mechanisms that have invoked an inactivation pathway originating at the compound II state (19, 36) are also not consistent with our data. No inactivation of the heme was observed when mCPBA reacted with an excess of compound II.
(i) The peroxidase inactivation pathway originates at a transient intermediate formed during the reaction of compound I with the hydroperoxide. This intermediate (P965) can be considered to be a model for the binding of reducing substrates to compound I and has spectral similarities to those of a complex formed between m-chlorobenzoic acid and compound I.
(ii) This intermediate decays in competing reactions to give compound II (67%) and an inactive form of HRP-C, P670 (33%) with mCPBA. The product ratio is determined only by the relative values of the inactivation and the catalytic rate constants.
(iii) The active center in different class III peroxidases appears to be well conserved (1), and the role of amino acids such as His-170, His-42, and Arg-38 is well established for HRP-C (11, 37). Apparently, this active center architecture is optimized for compound I formation, although with excess hydroperoxide inactivation occurs. Consequently, mutations in Arg-38 (22) or His-42 (37) make the variants more susceptible to inactivation by hydroperoxides. However, when conventional reducing substrates such as ABTS or guaiacol are present, the extent of inactivation is decreased since the steady-state level of compound I, which reacts with the hydroperoxide to initiate inactivation, is much lower, i.e. reducing substrate protects the enzyme from inactivation by removing compound I (38, 39).
(iv) It is interesting, from a physiological perspective, to point out that HRP-C shows a high affinity for hydroperoxides, shows a high rate of compound I formation, and is active at low concentrations of reducing substrate. However, it is more sensitive to inactivation under these conditions (21). These properties would allow HRP-C to act as an efficient detoxifying system for the elimination of excess hydroperoxide, providing the nature and concentration of reducing substrate ensures a competent rate of compound II reduction to ferric enzyme in the catalytic cycle. The danger of accumulating deleterious free radical could thus be avoided by a coupled process involving HRP reaction products and an antioxidant (i.e. ascorbic acid) (40, 41). However, if there is a perturbation in the cell status toward more oxidative conditions (i.e. the H2O2 burst induced by pathogenesis), then the detoxifying capacity of the enzyme could be surpassed, its susceptibility to inactivation would increase (at higher oxidant/reductant ratios), and the damaging free radical would not be reduced. Chain reactions could also result which can also contribute to cell death in the so-called "hypersensitive response" (42). The effect of H2O2 and mCPBA in vivo have recently been demonstrated by modifications in the patterns of growth and morphogenesis of lupin hypocotyls after application of different concentrations of these hydroperoxides (43).
The use of pre-steady-state kinetics under single turnover conditions has not only confirmed the values of the limited number of combined rate and equilibrium constants that we have previously determined under pseudo-steady-state conditions with mCPBA in large excess (26), but has also allowed the determination of the elementary rate constants for the partial reactions that comprise the mechanism of inactivation. In addition the UV-visible spectrum of the kinetically competent, common intermediate on the catalytic and inactivation pathways has been obtained. On the other hand, the topological similarity between mCPBA and commonly used suicide substrates such as phenylhydrazines (44, 45) and inhibitors like benzhydroxamic acid (24-26) make it a useful probe not only of aromatic substrate binding site(s) but also the heme access channel architecture and distal cavity, since it reacts with ferric enzyme to give compound I. Clearly, the characterization of mutants of HRP-C and naturally occurring isoenzymes such as HRP-A2 using mCPBA will provide not only additional mechanistic information but also valuable insights into the factors that determine the functional stability of peroxidases with commercial potential.
The proposed mechanism for the reaction of compound I with mCPBA is depicted in Scheme III (shown in Table I). This mechanism is a modification of a two-step mechanism (35) in which compound I is stable but mCPBA induces instability of the complex compound I-mCPBA (46). The time-course equations of the concentrations of compound II and inactive enzyme (P670) under single turnover are given for the following expressions.
![]() |
(Eq. 1A) |
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![]() |
(Eq. 2A) |
![]() |
(Eq. 3A) |
![]() |
(Eq. 4A) |
![]() |
(Eq. 5A) |
![]() |
(Eq. 6A) |
![]() |
(Eq. 7A) |
![]() |
(Eq. 8A) |
![]() |
(Eq. 9A) |
![]() |
(Eq. 10A) |
![]() |
(Eq. 11A) |
![]() |
(Eq. 12A) |
![]() |
(Eq. 13A) |
![]() |
(Eq. 14A) |
![]() |
(Eq. 15A) |
Therefore, the real time courses for compound II and P670 are given by Equations 16A and 17A.
![]() |
(Eq. 16A) |
![]() |
(Eq. 17A) |
A similar procedure can be followed to obtain the equation for
the reaction between compound II and mCPBA. In this case the first
order rate constants are named 1 and
2,
respectively (see Equations 9 and 10, under "Results and
Discussion").