(Received for publication, March 17, 1997, and in revised form, May 2, 1997)
From the Department of Biochemistry, Agricultural University Wageningen, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
The catalytic mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol by the covalent flavoprotein vanillyl-alcohol oxidase was studied. Using H218O, it was found that the carbonylic oxygen atom from the product 4-hydroxybenzaldehyde originates from a water molecule. Oxidation of vanillyl alcohol did not result in any incorporation of 18O.
Enzyme-monitored turnover experiments revealed that for both substrates
a process involving flavin reduction is rate determining. During
anaerobic reduction of vanillyl-alcohol oxidase by
4-(methoxymethyl)phenol, a relatively stable spectral intermediate
is formed. Deconvolution of its spectral characteristics showed a
typical pH-independent absorption maximum at 364 nm
(364 nm = 46 mM
1
cm
1). A similar transient species was observed upon
anaerobic reduction by vanillyl alcohol.
The rate of flavin reduction and synchronous intermediate formation by
4-(methoxymethyl)phenol is 3.3 s1 and is fast enough to
account for turnover (3.1 s
1). The anaerobic decay of the
intermediate was too slow (0.01 s
1) to be of catalytical
relevance. The reduced binary complex is rapidly reoxidized (1.5 × 105 M
1 s
1) and
is accompanied with formation and release of product. Oxidation of
free-reduced enzyme is an even faster process (3.1 × 105 M
1 s
1).
The kinetic data for the oxidative demethylation of 4-(methoxymethyl)phenol are in accordance with a ternary complex mechanism in which the reduction rate is rate-limiting. It is proposed that, upon reduction, a binary complex is produced composed of the p-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme.
Vanillyl-alcohol oxidase (VAO,1 EC
1.1.3.13) from Penicillium simplicissimum is a novel
flavoprotein that acts on a wide range of 4-hydroxybenzylic
compounds (1, 2). VAO is a homo-octamer, with each subunit containing
8-(N3-histidyl)-FAD as a prosthetic
group (3). During catalysis, the flavin cofactor is first reduced and
subsequently reoxidized by molecular oxygen to yield hydrogen peroxide.
In addition to the oxidation of aromatic alcohols also, demethylation,
deamination, and hydroxylation reactions are being catalyzed as shown
in Equation 1.
By its versatile catalytic potential, VAO may develop as a useful biocatalyst for applications in the fine chemical industry (4).
VAO is readily induced in P. simplicissimum by growth on veratryl alcohol (3). Although the enzyme is produced in relatively high amounts, the physiological role of the enzyme remained obscure for some time as VAO is not involved in the degradation of this aromatic alcohol. Only recently, it was found that the VAO-mediated oxidative demethylation of 4-(methoxymethyl)phenol is of metabolic relevance (5). When P. simplicissimum is grown on this phenolic methylether, VAO is induced and catalyzes the first step of the degradation pathway of 4-(methoxymethyl)phenol. Furthermore, analogs of 4-(methoxymethyl)phenol can easily be envisaged as physiological substrates enabling this ascomycetous fungus to cope with a wide variety of lignin decomposition products (5).
Previous studies have revealed some interesting mechanistic properties of VAO. A striking feature of all substrates is the necessity of a p-hydroxyl group that is probably a prerequisite for binding. Moreover, a large pKa shift observed upon binding of the competitive inhibitor isoeugenol indicates that substrates become deprotonated upon binding (1). For the reaction of VAO with the substrate eugenol, it was established that the oxygen atom incorporated into the formed product coniferyl alcohol is derived from water. From these results, a catalytic mechanism for the hydroxylation of eugenol was proposed which involves formation of a p-quinone methide intermediate (1). A similar catalytic mechanism has been proposed for the hydroxylation of 4-alkylphenols by the flavocytochrome, p-cresol methylhydroxylase (6). So far, no real evidence has ever been presented for the formation of p-quinone methide intermediates during flavin-mediated reactions. We have suggested that hydride transfer to the oxidized flavin cofactor following deprotonation of the substrate would be a feasible sequence of reactions leading to the formation of the labile p-quinone methide intermediate (1). Hydride transfer mechanisms have been proposed for several other flavin-dependent oxidases like methanol oxidase (7) and cholesterol oxidase (8). Recently, Mattevi et al. (9) provided evidence from crystallographic studies that, for D-amino acid oxidase also, a hydrid transfer is a likely event during catalysis. Because well diffracting crystals of VAO have been obtained, we aim to relate the catalytic properties of this flavoenzyme with the crystal structure in the near future (10).
In this paper, we report on the kinetic and catalytic mechanism of VAO
with the physiological substrate 4-(methoxymethyl)phenol. Evidence from
rapid reaction studies is presented which shows that the hydroxylation
of phenolic compounds by VAO involves the formation of a
p-quinone methide intermediate. Vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol; R1OCH3,
R2
H, and R3
OH in Equation 1) was included
as a model substrate in this study to examine both an oxidative
demethylation and an alcohol oxidation reaction.
VAO was purified from P. simplicissimum as described by De Jong et al. (3) with the modification that a 200-liter fermentor was used for cultivation and that cells were disrupted using a Manton-Gaulin homogenizer. The ratio A280/A439 for the purified enzyme was 11.0. Glucose oxidase (grade II) and catalase were from Boehringer Mannheim. H218O (97 mol/100 mol 18O) was obtained from Campro (Veenendaal, The Netherlands). Vanillyl alcohol, vanillin (4-hydroxy-3-methoxybenzaldehyde), 4-hydroxybenzaldehyde, and 4(methoxymethyl)phenol were purchased from Aldrich.
Analytical MethodsAll experiments were performed at
25 °C in 50 mM phosphate buffer, pH 7.5, unless stated
otherwise. VAO concentrations were calculated from the molar absorption
coefficient of the oxidized form (439 nm = 12.5 mM
1 cm
1 (3)).
For 18O incorporation experiments, 195 µl of H218O was added to 400 µl of 1.0 mM substrate solutions. After addition of VAO (25 µl, 200 µM) and catalase (5 µl, 100 µM), the samples were incubated for 5 min at 25 °C and subsequently twice extracted with 500 µl diethylether. After evaporation, the samples were analyzed by GC/MS. GC/MS analysis was performed on a Hewlett Packard HP 5973 MSD and HP 6090 GC equipped with an HP-5 column.
Steady-state KineticsSteady-state kinetic experiments were
performed essentially as described earlier (1). Vanillyl alcohol and
4-(methoxymethyl)phenol activity were determined spectrophotometrically
by recording the formation of vanillin (340 nm (pH 7.5) = 14.0 mM
1 cm
1) and
4-hydroxybenzaldehyde (
340 nm (pH 7.5) = 10.0 mM
1 cm
1), respectively. Oxygen
concentrations were varied by mixing buffers saturated with 100%
nitrogen and 100% oxygen in different ratios.
Stopped-flow kinetics were carried out with a Hi-Tech SF-51 apparatus equipped with a Hi-Tech M300 monochromator diode-array detector (Salisbury, United Kingdom). Spectral scans were collected each 10 ms. For accurate estimation of rate constants, single wavelength kinetic traces were recorded at 439 nm using a Hi-Tech SU-40 spectrophotometer. In anaerobic experiments, solutions were flushed with argon and contained glucose (10 mM) and glucose oxidase (0.1 µM) to ensure anaerobic conditions. To determine the maximal rate of enzyme reduction by 4-(methoxymethyl)phenol and vanillyl alcohol, apparent rates were determined at five different substrate concentrations. To obtain accurate estimations of reduction rate constants observed during anaerobic reduction by vanillyl-alcohol, measurements were also performed at 355 and 393 nm. Deconvolution analysis of spectral data was performed using the Specfit Global Analysis program Version 2.10 (Spectrum Software Assn., Chapel Hill, NC). Solutions containing reduced enzyme (5 µM) were prepared by titrating argon flushed enzyme solutions with dithionite. For generation of the reduced enzyme intermediate complex, the enzyme was anaerobically mixed with a 1.5-fold excess of 4-(methoxymethyl)phenol. Reoxidation of reduced enzyme was measured by monitoring the increase in absorption at 439 nm after mixing with molecular oxygen. Reduced enzyme (5.0 µM) was mixed with varying concentrations of molecular oxygen (10, 21, 50, and 100% saturation) to determine the second-order rate constants for the reoxidation of protein-bound flavin.
For enzyme-monitored turnover experiments (11), air-saturated enzyme and substrate solutions were mixed in the stopped-flow instrument after which the redox state of the flavin cofactor was recorded at 439 nm.
In a previous report, we already
identified the products formed from 4-(methoxymethyl)phenol and
vanillyl alchohol as their corresponding aldehydes (1). In this study,
H218O was used to test the involvement of water
in the VAO-mediated conversion of 4-(methoxymethyl)phenol and
vanillyl alcohol. Substrate solutions (containing 30%
H218O (w/w)) were incubated for 5 min in the
presence of a catalytical amount of enzyme. Blank reactions with the
aromatic products 4-hydroxybenzaldehyde and vanillin revealed that,
under these conditions, less than 5% of the carbonylic oxygen atoms
had exchanged with H218O. For the VAO-mediated
conversion of 4-(methoxymethyl)phenol, it was found that the aromatic
product 4-hydroxybenzaldehyde was fully hydroxylated by action of water
(97 ± 4%) (Fig. 1). The incorporation of
18O confirms an earlier finding (1) that conversion of
4-(methoxymethyl)phenol by VAO results in cleavage of the methoxyl
group as methanol. The VAO catalyzed oxidation of vanillyl alcohol to
vanillin did not result in significant 18O incorporation
(Fig. 1). This shows that with this alcoholic substrate water is not
involved in the enzymatic reaction.
Steady-state Kinetics
By measuring the VAO activity upon
varying the concentration of oxygen at different
4-(methoxymethyl)phenol concentrations, a set of parallel
Lineweaver-Burk plots was obtained. This suggests that for this
reaction a ping-pong mechanism may be operative. Parallel line kinetics
can, however, also occur in some limited cases of a ternary complex
mechanism where some specific rate constants are relatively small (12,
13). Fig. 2 shows a secondary plot of the extrapolated
turnover rates at saturating oxygen concentrations versus
the concentration of 4-(methoxymethyl)phenol. From this, the
steady-state kinetic parameters with 4-(methoxymethyl)phenol could be
calculated (Table I). The steady-state kinetic
parameters for vanillyl alcohol were similarly determined (again
showing series of parallel secondary plots) and were in the same range as for 4-(methoxymethyl)phenol (kcat = 3.3 s1, Km,S = 160 µM, Km,O2 = 28 µM). The relatively high Km value for
vanillyl alcohol might result from the more polar character of the
benzylic moiety compared with 4-(methoxymethyl)phenol (1).
|
By measuring the redox state of the flavin cofactor during catalysis
(enzyme-monitored turnover), information can be obtained about the
rate-limiting step (11). For this, the enzyme was aerobically mixed in
the stopped-flow apparatus with a high concentration of substrate. It
should be noted here that due to the low solubility of vanillyl alcohol
and 4-(methoxymethyl)phenol, the substrate concentrations (500 µM) were not fully saturating. During turnover, the
absorbance at 439 nm was monitored to detect the amount of oxidized
enzyme present. Fig. 3 shows that with both substrates most of the enzyme is in the oxidized state during turnover. The fraction of oxidized enzyme for both substrates was almost identical, 0.86 for 4-(methoxymethyl)phenol and 0.91 for vanillyl alcohol (Fig.
3). This suggests that processes involving flavin reduction are slower
than those of the oxidative part of the catalytic cycle.
Reductive Half-reaction
To study the reductive half-reaction
of VAO, the oxidized enzyme was mixed with substrate in the
stopped-flow spectrophotometer under anaerobic conditions. Reduction of
VAO by 4-(methoxymethyl)phenol was a monophasic process when monitored
at 439 nm. Diode-array detection revealed that anaerobic enzymatic
reaction with 4-(methoxymethyl)phenol resulted in the formation of a
species with an intense absorption maximum at 364 nm
(364 nm = 46 mM
1
cm
1) (Fig. 4). During this process, the
flavin becomes fully reduced as evidenced by the decrease in absorbance
at 439 nm. This indicates that the rate of the reverse reaction must be
relatively small. The rate of flavin reduction at saturated substrate
concentrations was in the same range as the turnover rate (Table I).
pH-dependent anaerobic reductions by
4(methoxymethyl)phenol revealed that the spectral properties of the
formed intermediate were not influenced between pH 6.8 and 7.9. Furthermore, reduction at the tested pH values did not result in a
significant change of the rate of reduction. When the spectral changes
were followed on a longer time scale (>20 s), a very slow decay of the
high absorbance intermediate was observed. The resulting spectrum could
be characterized as the composite of reduced enzyme and the product
4-hydroxybenzaldehyde. Indicative for aldehyde formation was the
increase in absorbance around 335 nm, which was even more pronounced at
higher pH due to deprotonation of the phenolic group
(pKa 4-hydroxybenzaldehyde = 7.6). The rate of
aldehyde formation under anaerobic conditions was estimated to be 0.01 s
1, which is too slow to be of catalytical relevance. The
results obtained from the anaerobic reduction of VAO with
4-(methoxymethyl)phenol are consistent with the following Equation 2,
![]() |
(Eq. 2) |
Reduction of VAO by vanillyl alcohol showed biphasic absorption traces
at 439 nm. Diode array analysis revealed that during reduction an
intermediate spectrum is formed with a typical absorption maximum at
362 nm (Fig. 5, A and B). As can
be seen from Fig. 5A, this intermediate decayed rapidly to
yield fully reduced enzyme. For accurate kinetic data, rate constants
of both reductive phases were determined at various substrate
concentrations by monitoring the reaction at isosbestic points (393 and
355 nm, see Fig. 5B). It was found that the first rapid
phase was an order of magnitude faster compared with the slow phase
(kred1 = 24 s1,
Kd = 270 µM compared with
kred2 = 3.5 s
1,
Kd = 150 µM; Fig. 6).
Using a consecutive irreversible reaction model in which the first
reaction corresponds to the fast process, spectral deconvolution
produced well defined spectra for the initial, intermediate, and final
components (Fig. 5B, traces A,
B, and C). As can be seen from Fig. 5B
the distinctive absorption maximum of the intermediate spectrum formed
in the first phase showed some resemblance with the intermediate
spectrum formed by anaerobic reduction of VAO with
4-(methoxymethyl)phenol (see Fig. 4 inset). This analogy was
confirmed by performing the anaerobic reduction experiments with
vanillyl alcohol at different pH values (Fig. 5B). As with
4-(methoxymethyl)phenol, no significant effect of pH on the observed
reduction rates was observed. Furthermore, these studies again showed
that in contrast to the transient intermediate spectrum, the final
spectrum is pH-dependent. This indicates that the final
product vanillin is formed only in the second step as the spectral
properties of the final spectrum agree nicely with formation of
vanillin (pKa = 7.5,
max = 345 nm).
Furthermore, in the case of vanillyl alcohol, the flavin apparently is
only partially reduced when the high absorbance intermediate is formed as the absorbance at 439 nm is relatively high after the first reductive phase (Fig. 5B).
Oxidative Half-reaction
To measure the rate of reoxidation,
reduced VAO was mixed with molecular oxygen in the stopped-flow
spectrophotometer, and the increase of the flavin absorbance at 439 nm
was monitored. Reoxidation of free-reduced VAO was a monophasic
reaction resulting in formation of fully oxidized enzyme. By varying
the concentration of oxygen, it was found that reoxidation of
free-reduced VAO is a fast bimolecular process (3.1 ×105
M1 s
1) as has been found for
other flavoprotein oxidases (14).
![]() |
(Eq. 3) |
As reduction by 4-(methoxymethyl)phenol resulted in a relatively stable
reduced enzyme-intermediate complex (Ered
~ Q), the rate of reoxidation of this complex was measured
as well. Therefore, after reducing VAO by 4-(methoxymethyl)phenol, the
reduced complex was mixed in the stopped-flow apparatus with oxygen to
measure the rate of formation of oxidized enzyme. It was found that the complex readily reacted with oxygen in a fast monophasic process reoxidizing the flavin with simultaneous product formation as evidenced
by the increase of absorbance at 335 nm. By varying the oxygen
concentration, the second order rate constant for reoxidation of the
reduced enzyme intermediate complex was estimated to be 1.5 × 105 M1 s
1. Similar
values for the rate of reoxidation have been reported for other
flavin-dependent oxidases (15-18). The apparent turnover rate with 4-(methoxymethyl)phenol during steady-state conditions is
much slower as the rate of reoxidation. Evidently, reoxidation is not
determining the rate of catalysis in case of 4-(methoxymethyl)phenol, which is in agreement with the enzyme monitored turnover results.
The experiments described here represent the first study on the
kinetic mechanism of VAO catalyzed reactions. Furthermore, evidence is
presented for the participation of p-quinone methides in the
catalytic mechanism of VAO. Previously, we proposed a reaction mechanism for the conversion of eugenol by VAO, which included formation of a p-quinone methide intermediate (1). Addition of water to this putative electrophilic intermediate would result in
the formation of the product coniferyl alcohol. From isotopic labeling
experiments, we have demonstrated in the present study the involvement
of water during the VAO catalyzed demethylation of
4-(methoxymethyl)phenol. VAO-mediated demethylation of this physiological substrate resulted in the introduction of an oxygen atom
originating from a water molecule. This indicates that during catalysis
the substrate is activated, after which water can attack the
C-atom. In contrast, with vanillyl alcohol, no oxygen atom derived from water is introduced into the aromatic product vanillin.
The rapid reaction data presented in this paper showed the formation of
intermediate reduced enzyme complexes during the reductive half-reaction. Anaerobic reduction of VAO by 4-(methoxymethyl)phenol revealed the formation of an eminently stable intermediate with typical
spectral properties (364 nm = 46 mM
1 cm
1). The spectral
characterisctics of the intermediate binary complex did not resemble
any known flavoprotein oxidase complex. Intermediate complexes formed
during catalysis of, for example, lactate monoxygenase (19) and
D-amino acid oxidase (20) have specific absorbances above
500 nm and are due to a charge transfer interaction between the reduced
enzyme and product. The formation of a flavin adduct as intermediate in
the reaction of VAO with 4-(methoxymethyl)phenol is rather unlikely as
the spectral properties of the intermediate complex do not resemble any
known flavin adduct spectrum (21). However, the spectral properties of
the formed intermediate generated during the anaerobic reaction of VAO
with 4-(methoxymethyl)phenol closely resembled reported spectra of
several p-quinone methides of structural analogs of the
phenolic substrate (
max ~ 360 nm,
~ 40 mM
1) as obtained by chemical synthesis or
flash photolysis of the corresponding phenols (22-26). However,
spectra of the p-quinone methides of vanillyl alcohol and
4-(methoxymethyl)phenol have never been described. These compounds are
highly unstable because of the lack of an electron donating group to
stabilize the electrophilic methide carbon atom. The data presented
here for VAO-mediated conversion of 4-(methoxymethyl)phenol are all
consistent with formation of a p-quinone methide
intermediate, which subsequently will react with water. The
p-quinone methide formed from this substrate is highly
stabilized in the active site as long as the enzyme remains reduced.
This suggests that the active site of the reduced enzyme intermediate
complex is solvent inaccesible. Upon reoxidation of the flavin, the
p-quinone methide intermediate rapidly reacts with water,
indicating that during this process local structural changes occur
leading to a more solvent accessible active site. The
p-quinone methide is hydrated to form the unstable hemiacetal product of 4-(methoxymethyl)phenol, which decomposes rapidly
to give 4-hydroxybenzaldehyde (Equation 4). The presence of reduced
glutathione during turnover of 4-(methoxymethyl)phenol did not
influence the stoichiometric formation of the aldehyde product,
indicating that hydration of the formed p-quinone methide occurs in the enzyme active site.
With vanillyl alcohol also, a transient intermediate spectrum was
observed during anaerobic reduction with similar spectral characteristics as with 4-(methoxymethyl)phenol. However, with vanillyl
alcohol, only half of the flavin is reduced when the maximal amount of
the high absorbance intermediate is formed, indicating that the
intermediate is already decomposed when the enzyme is fully reduced.
When the decay of the intermediate during the second reductive phase is
acknowledged, the molar absorption coefficient of the intermediate may
well resemble that of the 4-(methoxymethyl)phenol p-quinone
methide intermediate. The instability of the formed vanillyl alcohol
p-quinone methide may well explain why during oxidation of
this alcohol water is not involved in the formation of the aldehyde
product. Decay of the initial intermediate formed may correspond to the
autocatalytic decomposition of the vanillyl alcohol
p-quinone methide leading to vanillin. The biphasic reduction can be interpreted to result from two reactive conformations of the enzyme of which one is able to reduce the flavin rapidly while
the other conformation is slow in reduction. In view of this, it is
worthy to note that VAO is an octamer composed of relatively stable
dimers (27). Another explanation for the partial reduction observed
with vanillyl alcohol is a reversible reduction of the flavin by
vanillyl alcohol, which results in an equilibrium of oxidized and
reduced enzyme in anaerobic reduction experiments. When
k2 k
2, the ratio of
oxidized and reduced enzyme will be 0.5, leading to an apparent partial
reduction as shown in Fig. 5. The second phase observed during the
reductive half-reaction might then represent product release, which
would also result in total reduction of the flavin. In that case, the
turnover rate for vanillyl alcohol oxidation would be determined by
product release, which has also been found for several other
flavoprotein oxidases (11, 13). However, analysis of the
substrate-dependent reduction rates revealed that the
reversible step of reduction must be very small
(k
2 < 0.5 s
1) and is too small
to explain the relatively high amount of oxidized enzyme present after
the first phase of flavin reduction. Furthermore, enzyme-monitored
turnover experiments showed that the enzyme is mainly in the oxidized
state (91%) during steady-state turnover. As a consequence, with
vanillyl alcohol, a reductive step is limiting the turnover rate
also.
From the rapid reaction kinetic parameters obtained in this study, it
can be concluded that VAO catalyzes the oxidative demethylation of
4-(methoxymethyl)phenol via a ternary complex mechanism. Also, the
parallel lines pattern of Lineweaver-Burk plots found for the
steady-state kinetics are in accordance with a ternary complex mechanism as k2 is negligibly small and
k3 is relatively large (Table I). Because
k5 is very small, only a small portion of enzyme
will react via a ping-pong mechanism as represented by the left cycle
in Scheme I. From single turnover experiments, it could
be deduced that the rate of flavin reduction, i.e. formation of the
binary complex (k2 = 3.3 s
1), is
by far the rate determining step in catalysis
(kcat = 3.1 s
1). The
enzyme-monitored turnover results are also consistent with the proposed
kinetic mechanism. When the formation of the Michaelis-Menten complex
is a relatively fast process (at infinite substrate concentrations), the ratio of enzyme in the oxidized state during steady-state can be
calculated by the following.
![]() |
(Eq. 5) |
Taken together, the kinetic data are consistent with a ternary complex mechanism including (right cycle of Scheme I) 1) formation of a Michaelis-Menten complex, 2) flavin reduction and synchronous formation of the reduced enzyme intermediate complex, 3) reoxidation of the reduced enzyme complex by molecular oxygen with the concomitant conversion of the intermediate to form the final product, and 4) product release completing the catalytic cycle. Although a ternary complex mechanism is operative with 4-(methoxymethyl)phenol, with vanillyl alcohol, the reaction may also follow a ping-pong mechanism (represented by the left cycle in Scheme I). With this substrate, the binary reduced enzyme intermediate complex readily decomposes to form product without prior reoxidation of the flavin. In both cases, the rate of substrate-mediated flavin reduction is mainly limiting the overall rate of catalysis.
From the results presented in this study, it can be concluded that VAO efficiently converts the transient formed p-quinone methides. Quinone methides are highly electrophilic compounds and are thought to be involved in several toxicological processes. Studies have shown that formation of analogous quinone methides can result in (cyto)toxic effects by forming covalent bonds with cellular nucleophiles like proteins or DNA (25, 28). Preliminary release of the reactive product intermediate formed by VAO would also result in spontaneous reactions with water or other nucleophiles and could, therefore in vivo, elicit possible deleterious effects. Clearly, efficient hydration of the p-quinone methide in the active site of the enzyme is a prerequisite for the microorganism to exclude potential toxic effects.
We thank Dr. N. C. M. Laane for critically reading the manuscript.