From the Martin-Luther-Universität
Halle-Wittenberg, Institut für Biochemie and Institut für
Biotechnologie, Kurt Mothes Strasse 3, D-06099 Halle, Germany, the
§ Universität Konstanz, Fakultät Biologie, PF
5560-M644, D-78434 Konstanz, Germany, and the
¶ Department of Molecular and Cell Biology, University
of California, Berkeley, California 94720
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ABSTRACT |
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The phosphate- and oxygen-dependent
pyruvate oxidase from Lactobacillus plantarum is a
homotetrameric enzyme that binds 1 FAD and 1 thiamine diphosphate per
subunit. A kinetic analysis of the partial reactions in the overall
oxidative conversion of pyruvate to acetyl phosphate and
CO2 shows an indirect activation of the thiamine
diphosphate by FAD that is mediated by the protein moiety. The rate
constant of the initial step, the deprotonation of C2-H of thiamine
diphosphate, increases 10-fold in the binary apoenzyme-thiamine
diphosphate complex to 102 s
1. Acceleration
of this step beyond the observed overall catalytic rate constant to 20 s
1 requires enzyme-bound FAD. FAD appears to bind in a
two-step mechanism. The primarily bound form allows formation of
hydroxyethylthiamine diphosphate but not the transfer of electrons from
this intermediate to O2. This intermediate form can be
mimicked using 5-deaza-FAD, which is inactive toward O2 but
active in an assay using 2,6-dichlorophenolindophenol as electron
acceptor. This analogue also promotes the rate constant of C2-H
dissociation of thiamine diphosphate in pyruvate oxidase beyond the
overall enzyme turnover. Formation of the catalytically competent
FAD-thiamine-pyruvate oxidase ternary complex requires a second step,
which was detected at low temperature.
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INTRODUCTION |
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Pyruvate oxidase
(POX)1 from
Lactobacillus plantarum (EC 1.2.3.3) is a homotetramer. Each
subunit (mass, 65.5 kDa) binds one FAD and one thiamine diphosphate
(ThDP) in the presence of Mn2+ or Mg2+.
In the presence of oxygen and phosphate, the POX holoenzyme catalyzes
the oxidative decarboxylation of pyruvate (1-3) according to the
equation CH3-COCOO+O2 + HPO4
2 + 2H+
CO2 + H2O2 + CH3-COOPO3H
. In the presence of
Mn2+, both coenzymes FAD and ThDP can form binary complexes
with the apoenzyme that are enzymatically inactive in the native
overall oxidation reaction (4).
The important catalytic steps in the POX reaction are: step 1, deprotonation of C2-H of ThDP, the initial step shared by all ThDP dependent enzymes; step 2, binding of pyruvate to the C2 atom of enzyme-bound ThDP; step 3, decarboxylation of pyruvate to hydroxyethyl-ThDP; step 4, oxidation of hydroxyethyl-ThDP by FAD; and step 5, reoxidation of reduced FAD by oxygen. To study the role of both coenzymes in POX catalysis, we have used four different approaches that selectively investigate some of the individual steps enumerated above: (a) In the initial reaction (step 1), the deprotonation of the C2-atom of the ThDP in POX holoenzyme as well as in the binary ThDP complex was studied using 1H NMR by following the C2-H/D exchange (5). (b) DCPIP is known to accept electrons from hydroxyethyl-ThDP in other ThDP-dependent enzymes (6, 7). Here it was used as an artificial acceptor to assess the mode of electron transfer to O2. Two alternative donor loci have to be considered, the ThDP adduct itself or FADH2, to which redox equivalents could be transferred from ThDP. We have also used this assay with POX in which normal FAD was substituted with the analogue 5-deaza FAD (5-dFAD), which is essentially incapable of carrying out rapid redox reactions and in particular does not react with O2. This system was expected to provide information about steps 4 and 5 above. (c) Formation of the final product H2O2 was followed directly using the peroxidase assay (8). (d) The latter method was correlated by direct measurements of the O2 tension. In addition the different activity assays were used to investigate the recombination of FAD with the binary apo-ThDP complex.
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MATERIALS AND METHODS |
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Chemicals and Proteins
5-dFAD was synthesized from 5-deazariboflavin (9) using the purified FAD-synthetase complex of Brevibacterium ammoniagenes (10). FAD was from Boehringer Mannheim, and ThDP and bovine liver catalase (2000 units/mg) (EC 1.11.1.6) were from Sigma. Nitrogen 4.0 was from Messer-Griesheim. All other chemicals were of analytical grade and from Merck or Boehringer Mannheim. Quartz double distilled water was used throughout.
Pretreatment of POX Holoenzyme
Highly purified pyruvate oxidase (10 units/mg) from L. plantarum (EC 1.2.3.3) was a generous gift from Boehringer
Mannheim. 10 mg of lyophilized enzyme were saturated with 100 µM FAD and 1 mM ThDP/Mn2+ in 1 ml
of 0.2 M potassium phosphate buffer, 20% glycerol, pH 6.0, for 3 h at 20 °C. Subsequently the enzyme solution was dialyzed against 2 × 200 ml of 0.2 M potassium phosphate, 20%
glycerol, pH 6.0, for 12 h at 4 °C. The absorption ratio of
278/250 was 2.0 ± 0.1.
Preparation of Apoenzyme
Apoenzyme was prepared according to a modified method first
established by Strittmatter (11) and Sedewitz et al. (1). Lyophilized POX (5 mg) was dissolved in 0.2 ml of 0.2 M
potassium phosphate buffer, 20% glycerol, pH 6.0. Subsequently 0.2 ml
of 3 M sodium bromide was added, and the solution was
gently shaken at 0 °C for 2 min. 1.6 ml of a 50% saturated ammonium
sulfate solution adjusted to pH 3.0 with sulfuric acid were added
dropwise under gentle stirring at 0 °C. The precipitated apoenzyme
was sedimented by 15 min of centrifugation at 40000 × g at 4 °C. The yellow supernatant, which contained FAD
and ThDP, was discarded, and the pellet was resuspended in 0.2 ml of
0.2 M potassium phosphate, 20% glycerol, pH 6.0. The
ammonium sulfate precipitation step was repeated to completely remove
the enzyme-bound coenzymes. The resuspended cofactor-free apoenzyme (1 ml) was dialyzed four times for 4 h against 250 ml of the same
buffer at 4 °C. Traces of insoluble material were separated by
centrifugation at 20000 × g for 10 min at 4 °C. The
clear solution was stored at 4 °C. The absorption ratio of
278/250 was 3.3 ± 0.1.
Determination of Protein Concentration
Protein concentrations were determined spectrophotometrically.
For the holoenzyme extinction coefficients of 278 = 1.65 cm2/mg or of
460 = 0.235 cm2/mg
were used. Concentration of the apoenzyme was calculated using an
extinction coefficient of
278 = 1.07 cm2/mg
(4).
Assays
Determination of the Proton/Deuterium Exchange-- The kinetics of H/D exchange of the C2-H of ThDP were measured by 1H NMR as described recently (5). The exchange reactions were initiated by mixing equal volumes of a sample solution containing 10 mg/ml apo-ThDP-POX, holoenzyme or 1 mM ThDP alone in 0.1 M sodium phosphate buffer, pH 6.0, with D2O in a quenched flow apparatus (model RQF-3, Kin Tek Althouse) at 4 °C. The given pH values are the respective pH meter readings. Incubation times were varied between 2 and 2000 ms. Experiments with longer times were performed by manual mixing. The exchange reactions were stopped by addition of a final concentration of 0.1 M hydrochloric acid and 5% trichloroacetic acid. This procedure also rapidly and completely denatures and precipitates the protein and releases the cofactors ThDP and FAD. After separation of the denatured protein by centrifugation, the 1H NMR spectra of the supernatant containing ThDP and FAD were recorded in a 5-mm NMR tube on a Bruker ARX 500-MHz NMR spectrometer. As shown in Fig. 1, the signals used for quantification of the H/D exchange at C2 of ThDP do not interfere with the signals of FAD. To obtain the exchange rate, the relative decay in integral intensity of the C2-H signal at 9.68 ppm was fitted to a pseudo first order reaction (Fig. 2). The signal of the C6' proton at 8.01 ppm was used as a nonexchanging internal standard.
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DCPIP Assay--
Reduction of DCPIP can be followed by a
decrease in absorbance at 600 nm. The assay buffer contained 40 mM pyruvate in 50 mM potassium phosphate, 10%
glycerol, pH 6.0. Activity measurements were usually performed at
25 °C but at were performed at 10 °C for the recombination
experiments. POX activity was calculated using 600 = 17.7 cm2/µmol.
Determination of H2O2
Production--
Conditions were as in the DCPIP assay. Formation of
4-(p-benzochinone-monoimino)-phenazone from the reaction of
H2O2 with 4-aminoantipyrine (2 mM)
and 2,4-dichlorophenolsulfonic acid (7 mM) in the presence
of horseradish peroxidase (4 units/ml) is followed
spectrophotometrically at 546 nm (8). For the calculation of the
specific POX activity 546 = 16.5 cm2/µmol
was used.
Determination of O2 Consumption-- Conditions were as in the DCPIP assay. Oxygen tension was measured with a temperature-controlled O2 electrode (Rank Brothers, Cambridge, UK). The reaction was started by addition of POX.
Determination of the Recombination Kinetics
To follow recombination of FAD with the binary apo-ThDP complex, 1 µM apoenzyme was preincubated with 1 mM ThDP/Mn2+ in 0.2 M potassium phosphate buffer, 20% glycerol, pH 6.0, for 1 h at 10 °C, and subsequently 100 µM FAD was added. At the indicated times, samples were withdrawn to determine the activity either with the DCPIP or the H2O2 or the O2 detecting assays. For the incubations at both 25 and 10 °C, the assay temperature was 10 °C.
Production of Assay Solutions with Different Oxygen Concentrations
The assay mixtures were purged with nitrogen 4.0 for various
times. The residual oxygen concentration was measured with an oxygen
electrode from Metra Me und Frequenztechnik (Radebeul, Germany).
Detection of the Reaction Product in the DCPIP Assay
This was achieved by 1H NMR spectroscopy. The reaction was carried out with a small excess of DCPIP (1 mM) with respect to pyruvate (0.75 mM). The POX concentration in the incubation mixture was 1 mg/ml. Reaction products were separated from protein by centrifugation with Amicon Microcon-10 filters for 15 min at 7000 × g at 4 °C. 1H NMR spectroscopy was performed on a Bruker ARX 500 NMR Spectrometer. To suppress the water signal, water presaturation was used.
Spectroscopy
UV-visible absorption was recorded with a Kontron Uvicon-941 double beam spectrophotometer and fluorescence spectroscopy with a Hitachi F-3010 fluorescence spectrophotometer.
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RESULTS |
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Kinetics of H/D Exchange at the C2 Atom of ThDP in POX--
The
formation of all reaction intermediates in the POX reaction, detectable
by different activity assays, requires the deprotonation of the
C2-H of ThDP. This is the initial and key reaction for all
ThDP-dependent enzymes. The low rate of this reaction in
the free coenzyme ThDP was measured by H/D exchange and would not allow
catalysis to proceed at the observed rate of the enzyme reaction (Table
I). In the POX holoenzyme this reaction
is accelerated by 4 orders of magnitude compared with that of free ThDP
(Table I). This exchange rate reflects not only the deprotonation rate at the C2-H of ThDP but also the required exchange of the base with the
solvent. Therefore the observed rate could be slower but never faster
than the microscopic deprotonation rate for the C2-H proton. The value
of the exchange rate, however, exceeds the catalytic rate for this
enzyme (kcat = 2 s1 at 4 °C).
In the binary apo-ThDP complex this exchange rate is very low (Table
I). Interestingly, the second substrate phosphate further increases the
rate of the H/D exchange in the holoenzyme
16-fold compared with
that measured in a phosphate-free buffer (Table I).
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Preparation and Some Properties of 5-dFAD-POX--
The binary
apo-ThDP complex binds 5-dFAD, an analogue of FAD that is unreactive
toward O2 but that is isoelectronic with the latter. The
process is accompanied by a biphasic decrease of the fluorescence
emission of the 5-deaza-isoalloxazine (phase 1, 70% of the total
amplitude with k1 = 0.018 s
1, and
phase 2,
30% with k2 = 0.0025 s
1, Fig. 3A). It
should be noted that the recombination of native FAD proceeds with
essentially the same rate constants (
67% with k1 = 0.022 s
1 and
33% with
k2 = 0.0025 s
1) using the
fluorescence decrease of FAD as a probe (Fig. 3A). Binding
of 5-dFAD can also be monitored by following the increase of activity
in the DCPIP assay that accompanies it (Fig. 3B). The rate
of this process (k = 0.0022 s
1) is
essentially the same as that obtained for the slow phase of the
fluorescence changes. This suggests that 5-dFAD binds by the same mode
as native FAD, and this is also corroborated by the activity
measurements detailed below. When bound to the binary apo-ThDP complex,
5-dFAD accelerates the H/D exchange at the C2-H of ThDP beyond the
overall catalytic rate (Table I).
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POX Catalysis in the Presence of DCPIP--
DCPIP can act as an
artificial electron acceptor in ThDP-dependent enzymes (6,
7, 13), and, e.g. in pyruvate decarboxylase from yeast (EC
4.1.1.1.), it reacts specifically with the -carbanion of
hydroxyethyl-ThDP (6, 7). In the POX reaction, DCPIP acts as an
artificial electron acceptor too. Importantly, the product is acetyl
phosphate, i.e. the same as obtained in the native reaction.
This was shown by 1H NMR spectroscopy, which gave a signal
for the product at the chemical shift observed for acetylphosphate (2.1 ppm). We have thus employed DCPIP to assess the question of whether
this intermediate is oxidized in the POX catalysis as well. In the
presence of saturating pyruvate (40 mM) and phosphate (42 mM) and an O2 concentration of 0.25 mM, the reaction with DCPIP has a Km
value of
20 µM. Because a nonspecific inactivation by
the reaction of DCPIP with sulfhydryl groups was observed with other
enzymes (7, 14, 15) and in order to verify whether this would apply
also in the present case, 0.25 µM POX was incubated in
the absence and presence of 40 µM DCPIP for 1 h, and
subsequently the activity was measured. No effect of the preincubation
was observed, and thus a nonspecific inactivation by DCPIP can be
excluded for POX. In a phosphate-free solution (same pH value and ionic
strength, complete DCPIP assay mixture) DCPIP was not reduced by POX.
The binary apo-ThDP complex similarly is inactive in the absence and presence of phosphate. Solely in the presence of enzyme-bound ThDP,
Mg2+, FAD, and phosphate, DCPIP is reduced efficiently in
the POX reaction. Notably, the kcat and
Km values obtained in the DCPIP assay are about
2-fold increased compared with those measured in the
H2O2 assay (Table
II).
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Kinetics of FAD Association Followed by Activity Measurements and
Effect of Temperature--
The binding of FAD to the binary POX-ThDP
complex was investigated at different temperatures using the assays
described under "Materials and Methods." At 25 °C the three
methods revealed essentially the same result (Fig.
5B). The data points could be
fitted to a monophasic, pseudo first order process with
k = 0.016 s1 (100 µM
FAD).
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Catalytic Properties of 5-dFAD-POX--
The ternary complex of
5-dFAD, ThDP, and POX is completely inactive in the
H2O2 detecting assay but active in the test
using DCPIP. The dependence of the rate in the latter assay from DCPIP concentration follows a saturation behavior with a
Km of 24 µM. At saturating
substrate concentrations, the 5-dFAD-POX has 0.4% the activity of
native enzyme (Fig. 6). The possibility that this 0.4% activity results from traces of unremoved FAD can be
ruled out because apoenzyme did not show any residual activity. Furthermore, the procedure for the synthesis of 5-dFAD (9, 10) excludes
production of any normal FAD. 5-dFAD containing holo-POX was completely
inactive in the H2O2 assay. Incubation of the
5-dFAD containing POX with FAD has no effect on the observed rate in
the DCPIP assay. Similarly, incubation of native POX with 5-dFAD does
not affect the POX activity. This indicates that no dissociation/exchange of both FAD and 5-dFAD from the ternary complex
occurs.
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DISCUSSION |
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The essential steps constituting the cycle catalyzed by POX from
L. plantarum are shown in Scheme
1. Deprotonation of the ThDP-C2-H is
mandatory for the nucleophilic attack of the coenzyme ThDP
C2 on the pyruvate carbonyl, which leads to formation of
the enzyme-bound intermediate hydroxyethyl-ThDP. The rate of this
deprotonation for free ThDP and in the binary apo-ThDP complex is too
slow by about 3 orders of magnitude to account for the POX reaction to proceed at its observed overall catalytic rate of 2 s
1 at
4 °C (Table I). In native holoenzyme, i.e. in the ternary complex POX-FAD-ThDP, the rate of C2-H deprotonation has a higher value
than kcat, but in the presence of phosphate a
further 16-fold increase of the C2-H dissociation rate can be observed
(Table I). H+ abstraction does not appear to be mediated by
a direct interaction of the FAD with C2-H of the enzyme-bound ThDP but
rather by interactions with functional groups of the protein, which are
operative only in the POX-FAD-ThDP ternary complex. In the crystal
structure the distance of the closest FAD atom to C2-H is 11 Å (16,
17). However, based on the structural homology of the ThDP binding site
to other ThDP enzymes, we assume that Glu59 is the residue
that mediates this activation in POX. In POX the crucial interaction
could be that of the ThDP-N1' with the conserved Glu59 (16,
17). This would be analogous to what is observed with pyruvate
decarboxylase and transketolase where the same type of interaction
occurs (5). This interpretation is consistent with the observation that
in the ternary complex with 5-dFAD the rate of the H/D exchange at C2-H
is accelerated by 3 orders of magnitude compared with the binary
apo-ThDP complex (Table I). The substantial increase in rate observed
in the presence of phosphate cannot be interpreted in molecular terms
at the present time and might be due to a variety of effects.
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The next important steps in catalysis are the formation of the
pyruvate-ThDP adduct, which is followed by its decarboxylation to form
the hydroxyethyl-ThDP as shown in Scheme 1. DCPIP can react
specifically and rapidly with the latter intermediate. In POX, DCPIP
reduction requires both coenzymes and the cosubstrate phosphate.
Therefore, the redox partner for DCPIP in the POX reaction could be
either FADH2 or hydroxyethyl-ThDP. Because no competition between DCPIP and O2 was observed (Fig. 4) and because the
kcat value for the DCPIP assay is 2-fold
increased compared with that determined with the
H2O2 detecting assay (Table II), we conclude that different steps in catalysis are observed with the DCPIP and the
H2O2 detecting assays and that DCPIP does not
react competitively with FADH2. It has been discussed (17)
that the distance between the cofactors ThDP and FAD exclude a direct
electron transfer from the C2 of hydroxyethyl-ThDP to N5 of the
isoalloxazine (16). More likely, oxidation occurs via transfer of two
single electrons to the isoalloxazine (16). We conclude that DCPIP
reacts with the enzyme-bound intermediate hydroxyethyl-ThDP. The
occurrence of further intermediates during the electron transfer to FAD
clearly cannot be excluded; an intermediate could be inferred from the requirement of FAD for the DCPIP reduction.
Further evidence for the proposed mode of reaction of DCPIP arises from the experiments with 5-dFAD. This analogue cannot transfer electrons to O2. As expected, the 5-dFAD containing holoenzyme is inactive in the H2O2 detecting assay but reduces DCPIP (Fig. 6). The observation that in the presence of 5-dFAD, only 0.4% activity is observed in the DCPIP assay, although the H/D exchange for the ThDP C2-hydrogen is fast, points to a strong deceleration of a later step in the formation of hydroxyethyl ThDP in the presence of this FAD analogue. As pointed out under "Results," it can be ruled out that traces of native FAD are responsible for the residual activity. In the absence of phosphate no DCPIP activity is observed. Although phosphate stimulates the C2-H exchange of the enzyme-bound ThDP (Table I), this effect cannot be responsible for the essential function of phosphate. It is likely that phosphate is a cosubstrate in the formation of the product acetyl phosphate in the DCPIP reaction.
The differences among the kinetics of FAD recombination with the binary apo-ThDP complex at 10 °C using the different assays (Fig. 5) suggest that the final catalytically competent complex is formed via at least one intermediate form. This is assumed to be a holoenzyme in which formation of hydroxyethyl-ThDP but not transfer of electrons to O2 is possible. At 25 °C the lag phase in the recombination kinetics is not observable. This is compatible with the occurrence of a second step with a high activation energy during FAD binding. The possibility that the peroxidase reaction in the H2O2 detecting assay is rate-limiting at low temperatures can be ruled out, because all measurements of the FAD recombination process at 10 and 25 °C were performed at 10 °C.
The crystal structure for POX (16) shows that the isoalloxazine ring of the FAD is surrounded by six aromatic amino acids. Along the N10-N5-axis, the planar structure of the isoalloxazine ring is bent by 15 ° along the side chain of valine 265, which increases the redox potential of FADH2 (17). We suggest a model in which FAD binds in a two-step mechanism. The first leads to an intermediate that is competent exclusively in the formation of hydroxyethyl-ThDP. For this step FAD can be substituted by 5-dFAD. The nature of the second, temperature-sensitive step is still elusive at present. It could represent structural changes of the FAD binding site followed by the distortion of the planar structure of the isoalloxazine ring. FADH2, exclusively bound in this final conformation, is capable of performing the natural reaction, the reduction of O2 to H2O2.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
and Cell Biology, 229 Stanley Hall, University of California, Berkeley,
CA 94720. Tel.: 510-642-7684; Fax: 510-643-9092.
1 The abbreviations used are: POX, pyruvate oxidase; ThDP, thiamine diphosphate; 5-dFAD, 5-carba-5-deaza-FAD; DCPIP, 2,6- dichlorophenolindophenol.
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REFERENCES |
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