Pre-steady-state data for formation of Compound I for H42A and H42E were consistent with a mechanism involvingaccumulation of a transient enzyme intermediate Kd followed by conversion of
Trang 1Role of histidine 42 in ascorbate peroxidase
Kinetic analysis of the H42A and H42E variants
Latesh Lad1, Martin Mewies1, Jaswir Basran2, Nigel S Scrutton2and Emma L Raven1
1
Department of Chemistry and2Department of Biochemistry, University of Leicester, UK
To examine the role of the distal His42 residue in the
cata-lytic mechanism of pea cytosolic ascorbate peroxidase, two
site-directed variants were prepared in which His42 was
replaced with alanine (H42A) or glutamic acid (H42E)
Electronic spectra of the ferric derivatives of H42A and
H42E (pH 7.0, l¼ 0.10M, 25.0C) revealed wavelength
maxima [kmax(nm): 397, 509, 540sh, 644 (H42A); 404, 516,
538sh, 639 (H42E)] consistent with a predominantly
five-co-ordinate high-spin iron The specific activity of H42E for
oxidation ofL-ascorbate (8.2 ± 0.3 UÆmg)1) was 30-fold
lower than that of the recombinant wild-type enzyme
(rAPX); the H42A variant was essentially inactive but
activity could be partially recovered by addition of
exogen-ous imidazoles The spectra of the Compound I
intermedi-ates of H42A [kmax(nm)¼ 403, 534, 575sh, 645] and H42E
[kmax(nm)¼ 404, 530, 573sh, 654] were similar to those of
rAPX Pre-steady-state data for formation of Compound I
for H42A and H42E were consistent with a mechanism
involvingaccumulation of a transient enzyme intermediate
(Kd) followed by conversion of this intermediate into
Compound I (k¢1) Values for k¢1and Kdwere, respectively, 4.3 ± 0.2 s)1and 30 ± 2.0 mM(H42A) and 28 ± 1.0 s)1 and 0.09 ± 0.01 mM (H42E) Photodiode array experi-ments for H42A revealed wavelength maxima for this intermediate at 401 nm, 522 nm and 643 nm, consistent with the formation of a transient [H42A–H2O2] species Rate constants for Compound I formation for H42A were independent of pH, but for rAPX and H42E were pH-dependent [pKa¼ 4.9 ± 0.1 (rAPX) and pKa¼ 6.7 ± 0.2 (H42E)] The results provide: (a) evidence that His42 is critical for Compound I formation in APX; (b) confirmation that titration of His42 controls Compound I formation and
an assig nment of the pKafor this group; (c) mechanistic and spectroscopic evidence for an intermediate before Com-pound I formation; (d) evidence that a glutamic acid residue
at position 42 can act as the acid–base catalyst in ascorbate peroxidase
Keywords: ascorbate peroxidase; Compound I; histidine 42
The plant peroxidase superfamily has been classified [1] into
three major categories: class I contains the enzymes of
prokaryotic origin, class II contains the fungal enzymes (e.g
manganese peroxidase, lignin peroxidase) and class III
contains the classical secretory peroxidases [e.g horseradish
peroxidase (HRP)] The most notable member of the class I
peroxidase subgroup is cytochrome c peroxidase (CcP),
which was first identified in 1940 [2] In spite of the fact that
CcP has some rather unusual features, most notably the
existence of a stable tryptophan radical duringcatalysis
[3–6] and the utilization of a large macromolecular substrate
(cytochrome c), it has been the subject of such intense
mechanistic, structural and spectroscopic scrutiny that it has become the benchmark against which all other peroxidases are measured
More recently, it has been possible to isolate and purify in good yields a second member of the class I peroxidase subgroup, ascorbate peroxidase (APX) [7,8] Ascorbate-dependent peroxidase activity was first reported in 1979 [9,10] and the enzyme catalyses the reduction of potentially damaging H2O2 in plants and algae using ascorbate as a source of reducingequivalents [11,12] APX was known from sequence comparisons [13] to contain the same active-site Trp residue (Trp179) as is used by CcP (Trp191) during catalysis With high-resolution structural information avail-able for the recombinant pea cytosolic enzyme (rAPX) [14] (Fig 1), APX has provided a new opportunity to reassess the functional properties of CcP and to determine whether it
is indeed representative of class I peroxidases As detailed functional information has emerged, however, it seems that APX has several rather curious features of its own, and, in some ways, more questions have been raised than answered (In fact, even the current classification of APX as a class I enzyme has been recently questioned [15].) For example, Trp179 in APX is not a necessary requirement for oxidation
of ascorbate [16] and there is general agreement from kinetic [17–19] and EPR data [20] that the initial product (Compound I) of the reaction of APX with H2O2 is a porphyrin p-cation intermediate and not a protein-based trytophan radical Equally intriguing is the existence of a
Correspondence to E L Raven, Department of Chemistry,
University of Leicester, University Road, Leicester LE1 7RH, UK.
Fax: + 44 (0)116 2523789, Tel.: + 44 (0)116 2522099,
E-mail: emma.raven@le.ac.uk
Abbreviations: APX, ascorbate peroxidase; pAPX, wild-type pea
cytosolic APX; rAPX, recombinant wild-type pea cytosolic APX;
H42A, a variant of rAPX in which His42 has been replaced
with alanine; H42E, a variant of rAPX in which His42 has been
replaced with glutamic acid; CcP, cytochrome c peroxidase; HRP,
horseradish peroxidase; sh , shoulder.
Enzymes: ascorbate peroxidase (EC 1.11.1.11); horseradish peroxidase
(EC 1.11.1.7); cytochrome c peroxidase (EC 1.11.1.5).
(Received 27 December 2001, revised 8 April 2002,
accepted 9 May 2002)
Trang 2potassium-bindingsite (not present in CcP), located 8 A˚
from the a-carbon of Trp179; the functional role of this site
(if, indeed, there is one) is not yet fully understood
Although steady-state kinetic analyses have been a fairly
prominent feature of most of the early literature on APX
(reviewed in [12]), pre-steady-state kinetic data have been
very much more limited, largely as a result of insufficient
quantities of enzyme, and only preliminary mechanistic
information is available [16–19,21,22] The enzyme operates
through a classical peroxidase mechanism in which the ferric
enzyme is oxidized by two electrons to a so-called
Compound I intermediate with concomitant release of
one molecule of water, followed by two successive
single-electron reductions of the intermediate by ascorbate (HS) to
regenerate ferric enzyme
APXþ H2O2 !k1 Compound Iþ H2O ð1Þ
Compound Iþ HS !k2
Compound IIþ S ð2Þ Compound IIþ HS !k3
APXþ Sþ H2O ð3Þ Although Eqn (1) is commonly written as a single step,
experimental [23–26] and theoretical [27–30] evidence
sug-gests that this is an over-simplification A more complex
mechanism, as first suggested by Poulos and Kraut [31] –
involvingbindingof neutral peroxide to the enzyme,
concomitant proton transfer from the bound peroxide to
the distal histidine residue, followed by O–O bond cleavage
and release of H2O – has been suggested (Scheme 1) Of
particular interest is the role of the distal histidine residue (His42 in APX), which has been proposed [31,32] to act as
an acid–base catalyst, by acceptinga proton from H2O2and releasingit subsequently as water Site-directed mutagenesis studies on CcP and HRP have provided evidence to support these predictions (reviewed in [33–37]) In the work reported here, we replaced the distal histidine residue of APX (Fig 1) with alanine and glutamic acid (H42A and H42E variants, respectively) The aims of the work were severalfold First,
to establish a definitive role for His42 in Compound I formation by replacingit with a residue that is not capable
of hydrogen bonding (H42A) and to examine whether other residues at this position are able to act as alternative acid– base catalysts (H42E) Secondly, to use these variants to provide information on the origin of the pH-dependent kinetic rate profile for Compound I formation [17] Finally,
as we anticipated that the replacement of His42 would probably generate variant enzymes that may well have altered kinetic properties, we sought to utilize these alter-ations in intimate mechanism to probe in more detail the formation of Compound I in APX As such, we present the first spectroscopic evidence for the nature of the interme-diate formed duringthe reaction of APX with H2O2
M A T E R I A L S A N D M E T H O D S
Materials
L-Ascorbic acid (Aldrich Chemical Co.), guaiacol, imida-zole, 1,2-dimethylimidazole (Sigma Chemical Co.) and the chemicals used for buffers (Fisher) were of the highest analytical grade (more than 99% purity) and used without further purification H2O2 solutions were freshly prepared
by dilution of a 30% (v/v) solution (BDH): exact concen-trations were determined usingthe published absorption coefficient (e240¼ 39.4M )1Æcm)1[38]) Aqueous solutions were prepared using water purified through an Elgastat Option 2 water purifier, which itself was fed with deionized water All pH measurements were made usinga Russell pH-electrode attached to a digital pH-meter (Radiometer Copenhagen, model PHM 93)
Mutagenesis and protein purification Site-directed mutagenesis was performed according to the QuikchangeTMprotocol (Stratagene Ltd, Cambridge, UK) Two complementary oligonucleotides encoding the desired mutation were synthesized and purified (PerkinElmer) For H42A, the primers were: 5¢-CGTTTGGCATGGGCT TCTGCTGGTAC-3¢ (forward primer) and 3¢-GCAAAC CGTACCCGAAGACGACCATG-5¢ (reverse primer) For H42E the primers were: 5¢-CGTTTGGCATGGGAATC TGCTGGTAC-3¢ (forward primer) and 3¢-GCAAACC GTACCCTTAGACGACCATG-5¢ (reverse primer) To confirm the identity of the transformants, overnight cultures containing100 lgÆmL)1ampicillin were incubated at 37C with vigorous shaking (250 r.p.m.) The plasmid DNA was isolated usingthe Hybaid mini-plasmid system and sequenced to confirm the desired mutation Automated fluorescent sequencing, using New England Biolabs pUC and malE primers, was performed by the Protein and Nucleic Acid Chemistry Laboratory, University of Leices-ter, on an Applied Biosystems 373-Stretch machine, and
Scheme 1 Proposed steps in the formation of Compound I The
mechanism depicts the neutral bound and anionic
peroxide-bound intermediates The distal histidine residue that acts as the acid–
base catalyst is indicated (B).
Fig 1 Active site of ascorbate peroxidase [14] Hydrogen bonds
(dotted lines) are indicated.
Trang 3sequence data were analysed usingthe program SeqED
(Applied Biosystems) Individual mutations were confirmed
by sequencingacross the whole rAPX-codinggene
Bacterial fermentation of cells and purification of rAPX
were carried out accordingto published procedures [7]
Enzyme purity was assessed by examination of the ASoret/
A280value; in all cases an ASoret/A280value > 1.9 for rAPX,
H42A and H42E was considered pure Enzyme purity was
additionally assessed usingSDS/PAGE, and the
prepara-tions were judged to be homogeneous by the observation of
a single band on a Coomassie Blue-stained reducing SDS/
polyacrylamide gel Enzyme concentrations (pH 7.0,
l¼ 0.10M, 25.0C) were determined usingthe pyridine
haemochromagen method [39]: absorption coefficients were
e403¼ 88 mM )1Æcm)1for rAPX [40], e397¼ 83 mM )1Æcm)1
for H42A and e404¼ 95 mM )1Æcm)1for H42E
UV/visible spectroscopy
Spectra were recorded usinga variable-slit Perkin-Elmer
Lambda 14 UV/visible spectrometer, linked to an Exacta
486D computer, and an Epsom-LQ-1060 printer
Tempera-ture was controlled (± 0.1C) usinga thermally jacketed
cell holder connected to a circulatingwater bath (Julabo
U3) and a water cooler (MK Refrigeration Limited), which
was operated in tandem
Mass spectrometry
Samples were analysed usinga Micromass Quattro BQ
(Tandem Quadrupole) electrospray mass spectrometer
Horse heart myoglobin (Sigma) was prepared as described
for rAPX below, and used to calibrate the spectrometer in
the range 600–1400 m/z Protein samples were introduced
into the instrument at a flow rate of 5 lLÆmin)1 Trace salt
was removed usinga Centricon-10 concentrator (Amicon)
and successive centrifugation and dilution with highly
purified water (Elgastat) Samples ( 2 mgÆmL)1, 20 lL)
were then diluted 10-fold with a solution of 50 : 50 (v/v)
acetonitrile/water containing0.1% acetic acid
Steady-state measurements
Stock solutions of L-ascorbic acid, guaiacol, H2O2 and
enzyme were prepared in sodium phosphate (l¼ 0.10M,
pH 7.0, 25.0C) Enzyme assays were performed in a 1-mL
quartz cuvette: various concentrations of substrate and
25 nMenzyme were preincubated for 3 min in buffer and
the reaction was initiated by the addition of H2O2
( 2.5 lL, 30 mM) to a final concentration of 0.1 mM
The wavelengths and absorption coefficients used for
various substrates were as follows: L-ascorbic acid,
e290¼ 2.8 mM )1Æcm)1[41]; guaiacol, e470¼ 22.6 mM )1Æcm)1
[42] Activities were determined by dividingthe change in
absorbance by the absorption coefficient of the substrate
Values for kcatwere calculated by dividingthe maximum
rate of activity (lM )1Æs)1) by the micromolar concentration
of enzyme; values for Kmwere determined by a fit of the
data to the Michaelis–Menten equation usinga nonlinear
regression analysis program (Grafit32 version 3.09b;
Erithacus Software Ltd) All reported values are the
mean of three independent assays Errors on kcatand Km
are estimated to ± 5% and ± 10%, respectively For
pH-dependent assays, a mixed sulfonic acid buffer system (l¼ 95–110 mMdependingon the exact pH) that buffered over the entire pH range was used; reactions were initiated
by the addition of H2O2 (to 0.10 mM) In these cases, [L-ascorbic acid]¼ 0.70 mM(rAPX) and 0.50 mM(H42E), [guaiacol]¼ 30 mM (rAPX) and 11 mM (H42E), and [enzyme]¼ 25 nM Specific activities ([enzyme]¼ 25 nM, sodium phosphate, pH 7.0, l¼ 0.10M, 25.0C) were calculated from initial slopes of activity measurements;
1 unit of activity is defined as the amount of enzyme that oxidizes 1 lmol substrate per minute (lmolÆmin)1Æmg)1) Transient-state kinetics
Transient-state kinetics were performed usinga SX.18 MV microvolume stopped-flow spectrophotometer (Applied Photophysics) fitted with a Neslab RTE200 circulating water bath (± 0.1C) Reported values of kobs are an average of at least three measurements All curve fitting was performed usingthe Grafit software package All data were analysed usingnonlinear least-squares regression analysis
on an Archimedes 410–1 microcomputer usingSpectra-kinetics software (Applied Photophysics) Pseudo-first-order rate constants for the formation of Compound I (k1,obs) were monitored at 403 nm (rAPX), 404 nm (H42E) and 397 nm (H42A), in single mixing mode by mixing enzyme (0.5–1.0 lM) with various concentrations of H2O2 Absorbance changes were independent of [H2O2]; observed changes in absorbance were 97–99% of the calculated values The pH-jump method was used to examine the pH-dependence of Compound I formation, to avoid enzyme instability problems below pH 5 and above pH 8.5 Enzyme samples were prepared in water, adjusted to pH 7 with trace amounts of phosphate buffer (5 mM, pH 8.0); H2O2 solutions were made up in buffers of twice the final concentration The buffers used were sodium phosphate in the pH range 5.5–8.5 (l¼ 0.20M), citrate-phosphate in the
pH range 4.0–6.0 (l¼ 0.20M) and carbonate buffer in the range 8.0–9.0 (l¼ 0.20M) The pH of the solution was measured after mixingto ensure consistency pH-dependent data were fitted to the Henderson–Hasselbach equation for
a single-proton process (Eqn 4):
k¼Aþ B 10
pHpK a
where A and B are the rate constants for Compound I formation at the extremes of acidic and basic pH, respect-ively, and k is the rate constant (either second-order, k1, for rAPX or limitingfirst-order, k¢1, for H42A/H42E) for Compound I formation Formation of Compound I in the presence of exogenous imidazole was carried out using single-wavelength mode (397 nm), where one syringe con-tained H42A (1 lM) and the other H2O2(0.5–35 mM) in the presence of either imidazole or 1,2-dimethylimidazole (20 mM) (relatively low concentrations of exogenous imi-dazole and a high buffer concentration were used to minimize the effect of fluctuatingimidazole levels on the ionic strength and pH and to prevent binding of the imidazole to the haem) Time-dependent spectra of the various reactions were obtained by multiple-wavelength stopped-flow spectroscopy usinga photodiode array detec-tor and - software (Applied Photophysics) Spectral
Trang 4deconvolution was performed by global analysis and
numerical integration methods using PROKIN software
(Applied Photophysics)
R E S U L T S
Mass spectrometry
The integrity of the variant proteins was examined to ensure
that post-translational modification of the protein had not
occurred Analysis of H42A and H42E (data not shown)
gave average masses for the apoproteins of 27126.9 ±
0.8 Da and 27185.1 ± 0.5 Da, respectively, in good
agree-ment with the calculated masses of 27126.74 Da (H42A)
and 27184.88 Da (H42E)
Electronic spectra
Electronic spectra of the ferric derivatives of H42A and
H42E (pH 7.0, l¼ 0.10M, 25.0C) are shown in Fig 2
Wavelength maxima (Table 1) for H42A and H42E were
found to be slightly different from those for rAPX, but are
consistent with a predominantly five-coordinate high-spin
iron In the presence of excess cyanide, spectra for H42A
and H42E were consistent with the formation of a low-spin
haem species, with absorption maxima [H42A–CN kmax
(nm) (e (mM )1Æcm)1))¼ 420 (102), 540, 574sh; H42E–CN
kmax(nm) (e (mM )1Æcm)1))¼ 420 (109), 540, 573sh] similar
to those for the correspondingderivative of rAPX [rAPX–
CN kmax(nm) (e (mM )1Æcm)1))¼ 419 (104), 539, 572sh] The
spectra of both ferric H42A and H42E were, on the other
hand, unaffected by the addition of either azide or fluoride,
suggesting that these (weak field) ligands do not bind to the
haem under these conditions
Steady-state kinetics The specific activity of rAPX for oxidation ofL-ascorbic acid (256 ± 6 UÆmg)1) is comparable to published data (411 UÆmg)1) for pAPX [8] The specific activity of H42E (8.2 ± 0.3 UÆmg)1) was 30-fold lower than that of rAPX Under conditions identical with those used for rAPX and H42E, H42A exhibited no activity with
L-ascorbic acid, although residual activity was detected (9.2 ± 0.3· 10)2UÆmg)1) when a higher enzyme concen-tration was used ([H42A]¼ 200 nM)
Steady-state data (kcat, Kmand the arithmetically calcu-lated selectivity coefficient, kcat/Km) for oxidation of
L-ascorbic acid and guaiacol by rAPX and H42E are shown
in Table 2 (The oxidation ofL-ascorbic acid by rAPX does not obey standard Michaelis kinetics [8,22,43] and, in this case, data were fitted to the Hill equation Oxidation of guaiacol by rAPX obeys Michaelis kinetics and data were fitted to the Michaelis–Menten equation The origin of the different concentration-dependencies for these two sub-strates is not known Oxidation of bothL-ascorbic acid and guaiacol by H42E was observed to obey Michaelis–Menten kinetics.) For both substrates, kcatvalues for H42E are 50-fold lower than for rAPX, with Kmvalues largely unaffected (about threefold lower for H42E) The H42A variant was inactive with bothL-ascorbic acid (above) and guaiacol The dependence of the rate of substrate oxidation (lMÆs)1) vs
pH yielded a pH optimum for rAPX at 7 for the oxidation of both L-ascorbic acid (pH optimum 7.0) and guaiacol (pH optimum 6.9) (data not shown) (the pH optimum forL-ascorbic acid is consistent with that reported previously for pAPX [8]) For H42E, the pH optimum is shifted by 1 pH unit for both L-ascorbic acid (pH optimum 8.1) and guaiacol (pH optimum 8.0)
Fig 2 UV/visible spectra of ferric rAPX (solid
line), H42A (dotted line) and H42E (dashed
line) The region 450–700 nm has been
multi-plied by a factor of five Sample conditions:
sodium phosphate, pH 7.0, l ¼ 0.10 M ,
25.0 C.
Table 1 Wavelength maxima (nm) and in parentheses absorption coefficients (m M )1 Æcm)1) for the ferric and Compound I derivatives of rAPX, H42A and H42E.
Fe III 403(88), 506, 540 sh , 636 397(83), 509, 540, 644 404(95), 516, 538 sh , 639 Compound I 404(59), 529, 583 sh , 650 403(62), 534, 575 sh , 645 404(66), 530, 573 sh , 654
Trang 5Pre-steady-state kinetics
Spectra of the transient Compound I intermediates of
H42A and H42E, formed by reaction of the ferric
deriva-tives with 10 equivalents of H2O2, were obtained by
photodiode array experiments These preliminary
experi-ments showed that Compound I formation for both
variants is much slower than for rAPX, reactions being
complete in less than 1000 s for H42A and 100 s for H42E,
compared with less than 300 ms for rAPX under identical
conditions Wavelength maxima and absorption coefficients
for the Compound I intermediate of H42A and H42E were
found to be similar to those of rAPX (Table 1) and to those
previously published for wild-type pAPX (kmax¼ 404 nm
[17]) For H42A and H42E, Compound I is surprisingly
stable, for up to 30 s, but does not spontaneously convert
into Compound II as is observed for rAPX Instead,
Compound I for both H42A and H42E slowly returns to
a spectrum with a slightly red-shifted Soret band, with
wavelength maxima at 405, 514, 550sh and 640 nm for
H42A and 406, 516, 552shand 638 nm for H42E
The dependence of the observed rate constant, k1,obs
(individual traces were monophasic in all cases), on the
concentration of H2O2 for H42A and H42E (Fig 3)
exhibits hyperbolic behaviour For rAPX (data not shown
and [21,22]) and pAPX [17], a linear dependence on [H2O2]
is observed; in this work, a second-order rate constant of
(6.1 ± 0.1)· 107
M )1Æs)1was derived for reaction of rAPX with H2O2 Saturation behaviour of the kind exhibited by
H42A and H42E is consistent with a mechanism involvinga
pre-equilibrium step which precedes Compound I
forma-tion (Eqns 5 and 6):
Eþ H2O2 *)Ka
X!k
0 1
(E¼ H42A or H42E) This mechanism predicts a linear
(first-order) dependence at low concentrations of peroxide
and a zero-order dependence at high concentrations An
expression for k1,obscan be derived (Eqn 7):
k1;obs¼ k
0 1
1þ Kd=½H2O2 ð7Þ where Kdis the dissociation constant of the bound complex
in Eqn (5) (Kd¼ 1/Ka) and k¢1is the limitingfirst-order rate
constant at high peroxide concentrations A fit of these data
for H42A and H42E to Eqn (7) (Fig 3) yields values for k¢1
and Kd of 4.3 ± 0.2 s)1 and 30 ± 2.0 mM, respectively
(H42A) and 28 ± 1.0 s)1 and 0.09 ± 0.01 mM,
respect-ively (H42E) These data pass through the origin, indicating
that the second step of the reaction is irreversible At low concentrations of peroxide, where a linear dependence is observed, it is possible to extract an approximate value for the second-order rate constant for reaction with H2O2[Eqn (5) where Kdis related to the microscopic second-order (ka) and first-order (kb) rate constants for this step (Kd¼ kb/
ka)]: values for ka of 84 ± 6M )1Æs)1 and 1.1 ± 0.2·
105M)1Æs)1were obtained for H42A and H42E, respectively The mechanistic scheme implicated by the above data suggested the accumulation of a reaction intermediate, the conversion of which to product was rate-limitingat high peroxide concentrations To examine the nature of this intermediate, photodiode array experiments were carried out for H42A (Fig 4) Intermediate spectra were obtained from a spectrally deconvoluted model: A fi B fi C, where A corresponds to ferric H42A, B corresponds to the intermediate (tentatively assigned as the [H42A–H2O2] complex, vide infra) and C corresponds to Compound I Wavelength maxima for the proposed intermediate were at
401 nm, 522 nm and 643 nm The model yielded rate constants for each step: k (A fi B) and k (B fi C) of
Fig 3 Dependence of k 1,obs on [H 2 O 2 ] concentration for the reaction of H42A (A) and H42E (B) with H 2 O 2 (sodium phosphate, pH 7.0
l ¼ 0.10 M , 5.0 °C, [H42A] ¼ 0.5 l M , [H42A] ¼ 0.5 l M ) Data were fitted usinga nonlinear least-squares fittingprocedure to Eqn (7).
Table 2 Values for k cat , K m and k cat /K m for the oxidation of L -ascorbic acid and guaiacol by rAPX and H42E (sodium phosphate, pH 7.0, l¼ 0.10 M ).
Enzyme k cat (s)1) K m (m M )
k cat /K m
(m M )1 Æs)1) k cat (s)1) K m (m M )
k cat /K m
(m M )1 Æs)1)
Trang 6147 s)1and 1.19 s)1, respectively ([H2O2]¼ 35 mM), which
is in approximate agreement with the limiting rate constant
of 4.3 s)1determined above The maximum concentration
of the intermediate under these conditions was 91% of the
initial enzyme concentration Under these experimental
conditions and in contrast with rAPX, there was no clear
isosbestic point for the conversion of ferric H42A into
Compound I, indicatingthat there are more than two
absorbingspecies in solution and therefore consistent with
the formation of an intermediate At 342 nm (Fig 4, inset),
the isosbestic point between ferric enzyme and the
interme-diate, the absorbance–time trace shows a lagphase before
Compound I formation, providingfurther evidence to
support the proposed intermediate; the filled circles in this
inset are simulated data points derived from the model and
show good agreement between the simulated and
experi-mental data For H42E, no intermediate complex could be detected under these conditions
The pH-dependences of the observed rate constants for Compound I formation for rAPX, H42A and H42E enzymes are shown in Fig 5 Nonlinear dependencies with zero intercepts on [H2O2] were observed at all pH values for H42A and H42E The use of acetate and nitrate buffers has been avoided to prevent anionic effects previously observed
in pH-dependent studies on HRP and CcP [44–46] Compound I spectra for each enzyme were identical at all pHs (data not shown) Determination of second-order (k1) and limitingfirst-order (k¢1) rate constants for rAPX and H42E, respectively, revealed pH-dependent behaviour for Compound I formation in both cases (Fig 5A,C) These data were fitted to a sing le-proton process (Eqn 4), and pKa values for rAPX and H42E of 4.9 ± 0.1 and 6.7 ± 0.2, respectively, were obtained In contrast, H42A exhibits no detectable pH-dependence for the rate constant for Com-pound I formation (k¢1) from pH 5.5–8.5 (Fig 5B) Recovery of H42A activity
Steady-state and pre-steady-state analyses of H42A indi-cated that the enzyme was essentially inactive Rate constants for Compound I formation for H42A in the presence of either imidazole or 1,2-dimethylimidazole (20 m ) were also determined (sodium phosphate,
Fig 4 Photodiode array spectra (sodium phosphate, pH 7.0,
l ¼ 0.10 M , 5.0 °C) for the reaction between H42A (1 l M ) and H 2 O 2
(35 m M ) The reaction was monitored over a time base of 2 s, and 100
spectra were recorded with a time interval of 20 ms between each scan.
Experimental data were fitted to an A fi B fi C model, using
PROKIN software Deconvoluted spectra from this analysis are shown
in the Soret (A) and visible (B) regions The solid line shows the
spectrum of ferric H42A; the dotted and dashed lines show the
inter-mediate spectra derived from the model, and represent the spectra of
the proposed [H42A–H 2 O 2 ] complex and Compound I, respectively.
(B) Inset: time course at 342 nm (d) Simulated data points derived
from the model calculated usingthe PROKIN software.
Fig 5 pH-dependence of Compound I formation for (A) rAPX, (B) H42A and (C) H42E The solid line for rAPX and H42E represents a fit
of the data to the Henderson–Hasselbach equation for a single proton process (Eqn 4); data for H42A were fitted to y ¼ c Conditions:
l ¼ 0.10 M , 5.0 C.
Trang 7l¼ 0.20M, 5.0C, pH 7.0, [H42A] ¼ 1 lM; Fig 6).
[Higher concentrations of imidazole were not
experiment-ally accessible for two main reasons: (a) high
concentra-tions of either imidazole led to large changes in both ionic
strength and solution pH; (b) binding of imidazole to the
iron is observed at higher concentrations, leading to
enzyme inhibition.] The spectrum of Compound I formed
under these conditions (kmax¼ 403, 534, 575sh, and
645 nm) was identical with that obtained in the absence
of exogenous imidazoles Formation of Compound I
again showed saturation kinetics (Fig 6), and a nonlinear
least-squares fit of the data to Eqn (7) in the
pres-ence of imidazole and 1,2-dimethylimidazole yields
k¢1¼ 40 ± 3 s)1 and 168 ± 30 s)1, respectively, and
Kd¼ 25 ± 5 mMand 23 ± 8 mM, respectively
Compar-ison with the correspondingdata obtained under similar
experimental conditions but in the absence of exogenous
ligand [k¢1¼ 4.1 ± 0.1 s)1, Kd¼ 30 ± 2.0 mM (sodium
phosphate, pH 7.0 l¼ 0.20M], indicates that imidazole
and 1,2-dimethylimidazole lead to 10-fold and 40-fold
increases in k¢1, respectively, with the values for Kdlargely
unaffected [These values are slightly different from
those determined above (k¢1¼ 4.3 ± 0.2 s)1 and
Kd¼ 30 ± 2.0 mM), because the two determinations were
carried out at a slightly different ionic strengths.]
In parallel steady-state experiments ([H42A]¼ 100 nM,
[guaiacol]¼ 30 mM, [H2O2]¼ 0.10 mM, sodium
phos-phate, pH 7.0, l¼ 0.20M, 25.0C), the oxidative activity
of H42A with guaiacol was observed to increase with
increasingconcentration of imidazole and to saturate at
high concentration of imidazole (data not shown)
(L-Ascorbic acid activity was not examined because both
L-ascorbic acid and imidazole absorb strongly between 265
and 290 nm.) Separate UV/visible experiments (data not
shown) provided independent evidence for bindingof
imidazole to the iron (Kd¼ 51 ± 6 mM for H42A;
Kd¼ 0.3 ± 0.1 mM for rAPX) to generate a low-spin
H42A–imidazole derivative (kmax¼ 412, 533 and 565shnm)
that inhibited activity Guaiacol activities in the presence of
1,2-dimethylimidazole were 10-fold higher than for imidazole itself and were observed to increase linearly with increasing1,2-dimethylimidazole concentration (1–40 mM);
no evidence for saturation was observed in this case and addition of 1,2-dimethylimidazole to H42A did not generate
an observable low-spin derivative
D I S C U S S I O N
To investigate the catalytic role of the conserved His42 residue in APX catalysis, two site-directed variants were prepared in which the distal histidine was replaced by alanine (H42A) and glutamic acid (H42E) residues The results provide: (a) unambiguous evidence that His42 is critical for efficient Compound I formation in APX; (b) confirmation that titration of this residue controls the rate constant for Compound I formation and an assignment of the pKa for this group; (c) mechanistic evidence for an intermediate before Compound I formation; (d) spectro-scopic evidence on the nature of this intermediate The detailed implications of these data are discussed below Examination of the electronic spectra for the two variants indicates that large-scale structural alterations have not been induced as a direct consequence of the mutations Hence, wavelength maxima for ferric H42A and H42E are shifted slightly compared with ferric rAPX (Table 1), but are consistent with a largely five-coordinate, high-spin haem geometry As ferric rAPX has itself been found [47] to be a mixture of five and six-coordinate high-spin haem together with some six-coordinate low-spin haem (the relative proportions of which are themselves likely to be affected
by sample and storage conditions), it is probable that the slight differences in the exact wavelength maxima reflect differences in the spin state/coordination number distribu-tion for each variant compared with rAPX In particular, we note the broad Soret band, distinct shoulder ( 375 nm) and red-shifted CT1 band (644 nm) for H42A, all of which are consistent with increased five-coordinate character for this variant compared with both rAPX and H42E How-ever, it is not possible to make fully quantitative assessments from these data, and more detailed spectroscopic analyses were not attempted [although resonance Raman data (unpublished work) for rAPX and H42A at neutral pH indicate that the five and six-coordinate high-spin haem represent major and minor components, respectively] The steady-state oxidation ofL-ascorbate by pAPX [8] and rAPX [43] cannot be satisfactorily fitted to a standard Michaelis–Menten treatment, and sigmoidal kinetics are observed; steady-state oxidation of guaiacol by rAPX obeys Michaelis–Menten kinetics Although it was initially pro-posed that the origin of the sigmoidal dependence may arise from the dimeric structure of the enzyme, site-directed mutagenesis work [43] has indicated that this is unlikely to
be the case It is possible that nonspecific effects, perhaps involvingradical chemistry associated with oxidation of ascorbate, or the existence of more than one substrate bindingsite, may be influential A sigmoidal dependence on substrate concentration is not observed for H42E-catalysed oxidation ofL-ascorbate, an observation that we are not, at present, able to fully rationalize Replacement of the distal His42 by alanine renders the H42A enzyme essentially inactive, which is presumably directly linked to the very poor reactivity of this variant with HO
Fig 6 Dependence of k 1,obs , the pseudo-first-order rate constant for
Compound I formation in H42A, on H 2 O 2 concentration in the absence
and presence of exogenous imidazoles (20 m M ) Conditions: sodium
phosphate, l ¼ 0.20 M , 5.0 C, pH 7.0, [H42A] ¼ 1 l M (n) H42A;
(s) H42A + imidazole; (d) H42A + 1,2-dimethylimidazole.
Trang 8There is now general agreement that the Compound I
species formed immediately after reaction of both rAPX
and pAPX with H2O2contains a porphyrin p-cation radical
and not a protein-based radical as observed in CcP [17–20]
Spectra of Compound I obtained in this work for rAPX
agree with those previously published for pAPX
(kmax¼ 404 nm [17]) and those for HRP in which a
porphyrin p-cation radical is known to be generated [37]
Spectra for the Compound I intermediates of H42A and
H42E were similar to those of rAPX and are also consistent
with a porphyrin p-cation formulation The decay of
Compound I of rAPX in the absence of substrate through
the normal Compound II/ferric route is not straightforward
and generates spectra that are similar to, but not identical
with, authentic samples of either Compound II or ferric
rAPX [48] It has been proposed [48] that (noncatalytic)
protein radical chemistry involvinga Trp amino acid occurs
under these conditions, leadingto a permanent alteration of
the haem structure that is reflected in the absorption
maxima of the final (ferric-like) decay product For H42A
and H42E in the absence of substrate, Compound I does
not spontaneously convert into a Compound II species
as observed for rAPX, and the Compound I intermediates
for both enzymes slowly generate species with a slightly
red-shifted Soret band [kmax (H42A)¼ 405 nm; kmax
(H42E)¼ 406 nm] compared with the ferric state These
maxima are analogous to those observed for the decay of
rAPX (kmax¼ 406 nm), suggesting that similar radical
chemistry may occur in the variants, although this has not
been examined in detail in this work The failure to observe
Compound II for the two variants under these conditions is
intriguing (particularly as H42E is clearly active against
both L-ascorbate and guaiacol) and has been noted
previously for the H42E [49,50] and H42A and H42V [51]
variants of HRP These data suggest that the distal histidine
residue stabilizes Compound II through a
hydrogen-bond-ingstructure involvingthe Ne of His42 and the ferryl
oxygen However, although hydrogen-bonding interactions
of this kind have been structurally confirmed for the
ferrous-oxy derivative of CcP [52], no such interaction was detected
in the crystal structure of Compound I [53,54] Instead,
stabilizinghydrogen-bondinginteractions from the distal
arginine residue have been identified for Compound I
[53,54] Indeed, Compound II of the R38A variant of
HRP was also not detected [25] and the Compounds I of the
R48L, R48K [55] and R48E [56] variants of CcP are
similarly unstable Hence, it is possible that these
hydrogen-bondinginteractions involvingArg38 in rAPX have been
simultaneously disrupted as a secondary consequence of the
His42 mutations and, together with His42, may be
influen-tial in definingthe stability of Compound II
The replacement of the distal histidine residue by alanine
or glutamic acid clearly has a profound influence on the
ability of the rAPX enzyme to react with H2O2 and is
clearly demonstrated by the nonlinear dependence of the
observed rate constant on peroxide concentration A
comparison of rate constants for rAPX and variant
proteins is only possible by comparingthe second-order
rate constant derived from the linear part of Fig 3 with
the second-order rate constant for formation of
Com-pound I in rAPX These values indicate that a 106-fold
and 102-fold decrease has occurred for H42A and H42E,
respectively These data provide convincingevidence to
support a key catalytic role for His42 and indicate that the glutamic acid residue is able to replace the distal histidine residue such that reasonably efficient Compound I forma-tion is effected The correspondingH42A and H42E variants in HRP have been shown to lower the rate constant for Compound I formation by six and four orders of magnitude, respectively [49–51,57], although no evidence for an intermediate was found for these variants Large effects on the rate constant of Compound I formation have also been reported for other His42 variants
of HRP [58–60] and for CcP [61,62]
The pre-steady-state data (Fig 3) are consistent with a mechanism involvingformation of an enzyme–substrate intermediate, the conversion of which into product is rate-limitingat high concentrations of peroxide The simplest mechanism that is consistent with these data is described by Eqns (5) and (6) Although mechanisms involving conform-ational gating of the reaction at high peroxide concentra-tions [55] or before Compound I formation [63] are possible, the observation of a spectroscopically distinct intermediate for H42A provides good evidence for the proposed mechanism (but leaves this interpretation slightly more open for H42E) This enzyme intermediate has wavelength maxima at 401 nm, 522 nm and 643 nm and
we assign the intermediate as arising from a transient [H42A–H2O2] species (see below) Our ability to detect this species for the first time arises from the loweringof the observed rate constant compared with rAPX as a conse-quence of the mutation, such that high concentrations of
H2O2 (above the Kd) are experimentally accessible in the stopped-flow experiment It seems unlikely that rAPX would utilize a different mechanism and we assume that detection of the intermediate is not possible in this case because the concentrations of H2O2required to satisfy the inequality [H2O2] d would generate observed rate constants outside of the experimental stopped-flow limit
As such, only the linear part of the nonlinear k1,obs vs [H2O2] dependence is experimentally accessible for rAPX, and O–O bond cleavage is not rate-limiting under any experimental conditions [In fact, for all APXs [17–19] and variants of rAPX [16,21,22] examined so far, a linear dependence on [H2O2] is observed and second-order rate constants are derived (k1 107
M )1Æs)1).] Indeed, this intermediate has eluded detection for this very reason in other peroxidase systems and its exact structure is still not clear For example, Baek & Van Wart [23,24] identified an intermediate, designated Compound 0 and proposed to be a hyperporphyrin (FeIII-OOH) complex, in the reaction of HRP with various peroxides under cryogenic conditions (kmax 330, 410 nm for HRP-H2O2) Transient interme-diates have also been detected for HRP in polyethylene glycol [26] and for the R38L/R38G and H42L variants of HRP [25,64] Comparison of the spectra obtained in this work with theoretical calculations [29] on the hyperporph-yrin (FeIII-OOH) vs neutral peroxide (FeIII-HOOH) struc-ture for this intermediate, together with the similarity of the spectrum of the intermediate to that of the ferric derivative, indicates that the new intermediate for H42A is probably a neutral ferric peroxide complex This suggests that the substrate is bound initially as a neutral peroxide, consistent with the known pKaof H2O2(pKa¼ 11.6), which dictates that the peroxide molecule is predominantly in the proto-nated form under the conditions used in this work
Trang 9(deprotonation of the bound hydroperoxide species is
assumed to occur before O–O bond cleavage)
The pH-dependent data for Compound I formation,
Fig 5, are particularly informative The absence of a
pH-dependence for H42A unambiguously assigns this residue as
beingresponsible for the pH-dependent reaction between
rAPX and H2O2 The pKa of His42 can also be derived
directly (pKa¼ 4.9) from the rAPX data and indicates that
this group must be deprotonated for efficient reaction with
H2O2(Scheme 1) By analogy with the rAPX data, the new
pKa observed for H42E (pKa¼ 6.7) can be assig ned as
arisingfrom titration of the glutamic acid residue at position
42: although this is slightly higher than the pKaof the free
amino acid (pKa¼ 4.1), it is well known that protein pKa
values are a sensitive function of the protein environment
and that protein structure is often able to modulate
thermodynamic proton-transfer events over a wide range
(for example as evidenced from the range of pKa values
exhibited by the distal residue in peroxidases [65]) The pKa
for His42 corresponds closely to that reported previously
(pKa¼ 5.0 [17]) for pAPX, although for the pAPX enzyme
no assignment of this titratable residue was possible For
peroxidases that exhibit pH-dependent kinetics of
Com-pound I formation, the pKavalues are in a similar range to
that found in this work (for example, HRP (pKa¼ 2.5–5.3
[66,67]), myeloperoxidase (pKa¼ 4.0 [68,69]) and Coprinus
cinereusperoxidase (pKa¼ 5.0 [70]) and have been assigned
as arisingfrom titration of the distal residue For
peroxid-ases that exhibit pH-independent kinetics, for example,
lignin [71] and manganese peroxidases [72], titration of the
distal histidine presumably still influences Compound I
formation, but the pKais not experimentally accessible For
CcP, examination of the role of the distal histidine residue in
Compound I formation has been complicated by specific
buffer effects that alter the kinetic profile [46,62] Where
pH-dependent rate constants have been reported, the pKavalues
(pKa¼ 5.4 [62], 4.0 [62]) are in a similar range to those
reported here for rAPX
The catalytic deficiency of H42A can be partially
com-pensated for by the use of exogenous imidazole [57]; we
observed 1,2-dimethylimidazole to be more effective than
imidazole in recoveringactivity against guaiacol This is
probably related to the relative affinities of the two imidazole
derivatives for bindingto the haem iron: bindingof imidazole
but not 1,2-dimethylimidazole is observed, leadingto a linear
dependence of the activity over the entire concentration
range for 1,2-dimethylimidazole, but not for imidazole The
effect of exogenous imidazoles is also evident for the rate
constant for Compound I formation for H42A, which was
enhanced by 10-fold and 40-fold for imidazole and
1,2-dimethylimidazole, respectively For both imidazoles,
how-ever, the rate constant for Compound I formation and the
peroxidase activity are still much lower than for rAPX itself,
clearly highlighting the very specific role that the distal acid–
base catalyst plays in peroxidase catalysis
A C K N O W L E D G E M E N T S
This work was supported by grants from the BBSRC (Special
Studentship to L L and project grant 91/B11469), the Royal Society
(grants 18851 and 21138) and Zeneca (CASE award to L L.) Mr
J Lamb and Professor Peter Farmer (Centre for Mechanisms of
Human Toxicity, Leicester University) are gratefully acknowledged for
assistance with MS analyses Drs Ian Ashworth and Brian Cox (Syngenta formerly Zeneca) are gratefully acknowledged for supporting this work We are also grateful to Mr Kuldip Singh for technical assistance.
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... able to fully rationalize Replacement of the distal His42 by alanine renders the H42A enzyme essentially inactive, which is presumably directly linked to the very poor reactivity of this variant... (particularly as H42E is clearly active againstboth L -ascorbate and guaiacol) and has been noted
previously for the H42E [49,50] and H42A and H42V [51]
variants of. .. HRP in polyethylene glycol [26] and for the R38L/R38G and H42L variants of HRP [25,64] Comparison of the spectra obtained in this work with theoretical calculations [29] on the hyperporph-yrin