From the Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210-1218
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ABSTRACT |
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The kinetics of xanthine oxidase has been
investigated with the aim of addressing several outstanding
questions concerning the reaction mechanism of the
enzyme. Steady-state and rapid kinetic studies with
the substrate 2,5-dihydroxybenzaldehyde demonstrated that
(kcat/Km)app
and kred/Kd exhibit
comparable bell-shaped pH dependence with pKa
values of 6.4 ± 0.2 and 8.4 ± 0.2, with the lower
pKa assigned to an active-site residue of xanthine oxidase (possibly Glu-1261, by analogy to Glu-869 in the
crystallographically known aldehyde oxidase from Desulfovibrio
gigas) and the higher pKa to substrate. Early
steps in the catalytic sequence have been investigated by following the
reaction of the oxidized enzyme with a second aldehyde substrate,
2-aminopteridine-6-aldehyde. The absence of a well defined acid limb in
this pH profile and other data indicate that this complex represents an
Eox·S rather than
Ered·P complex (i.e. no chemistry
requiring the active-site base has taken place in forming the long
wavelength-absorbing complex seen with this substrate). It appears that
xanthine oxidase (and by inference, the closely related aldehyde
oxidases) hydroxylates both aromatic heterocycles and aldehydes by a
mechanism involving base-assisted catalysis. Single-turnover
experiments following incorporation of 17O into the
molybdenum center of the enzyme demonstrated that a single oxygen atom
is incorporated at a site that gives rise to strong hyperfine coupling
to the unpaired electron spin of the metal in the MoV
oxidation state. By analogy to the hyperfine interactions seen in a
homologous series of molybdenum model compounds, we conclude that this
strongly coupled, catalytically labile site represents a
metal-coordinated hydroxide rather than the Mo=O group and that this
Mo-OH represents the oxygen that is incorporated into product in the
course of catalysis.
Xanthine oxidase from cow's milk is a homodimer with a molecular
mass of 300 kDa, with each catalytically independent subunit possessing
four prosthetic groups: one molybdenum center, one FAD, and two
Fe2S2(Cys)4 iron-sulfur centers.
Physiologically, the enzyme catalyzes the oxidative hydroxylation of
hypoxanthine to xanthine and the subsequent hydroxylation of xanthine
to uric acid, the final two steps of purine metabolism in mammals.
Substrate hydroxylation takes place at the molybdenum center of the
enzyme, which becomes reduced from MoVI to MoIV
in the process (1). The reducing equivalents introduced at the
molybdenum center are subsequently passed via intramolecular electron
transfer to the flavin center, where reaction with O2 takes
place to give peroxide or superoxide, depending on the level of enzyme
reduction (2, 3).
Xanthine oxidase exhibits an unusually broad specificity toward
reducing substrates. It can hydroxylate a wide variety of purines,
pteridines, and related aromatic heterocycles and also a range of both
aliphatic and aromatic aldehydes, taking these to the corresponding
carboxylic acid. Indeed, xanthine oxidase belongs to the same family of
mononuclear molybdenum enzymes (the molybdenum hydroxylases) as do the
aldehyde oxidases. These enzymes have similar cofactor constitutions
and amino acid sequences and also have substantially overlapping
specificity for reducing substrates (4). The specificity of xanthine
oxidase toward aldehyde substrates has been examined by Morpeth (5),
who has found that biogenic aldehydes such as acetaldehyde,
indole-3-aldehyde, pyridine-2-aldehyde, etc. are reasonable substrates
for the enzyme. To investigate the mechanism of aldehyde hydroxylation
by xanthine oxidase and related molybdenum hydroxylases, the reaction
of the enzyme with 2,5-dihydroxybenzaldehyde and
2-aminopteridine-6-aldehyde
(PTA)1 has been examined. The
pH dependence of the kinetics for these reactions indicates that, as
with purine hydroxylation, hydroxylation of aldehyde substrates takes
place via a base-catalyzed mechanism and substrate must be protonated
for hydroxylation to occur.
The reaction catalyzed by the molybdenum hydroxylases is unusual in
comparison with that of other biological hydroxylation systems in that
water rather than dioxygen is the ultimate source of the oxygen atom
incorporated into product (4), and the reaction generates rather than
consumes reducing equivalents. Although the oxygen atom incorporated
into product is ultimately derived from water, it has been known for
some time that the proximal oxygen atom donor is a catalytically labile
site on the enzyme, which, in the course of a single turnover,
transfers its oxygen to substrate, to be regenerated subsequently by
oxygen derived from solvent prior to a second turnover (6). In light of
the known precedence for oxo transfer in the literature of small
inorganic complexes of molybdenum (7-16), it was originally thought
most likely that the catalytically labile site of the enzyme was the Mo=O group. More recently, it has been suggested that the catalytically labile site might instead be a metal-coordinated hydroxide (17-19). Given the crystallographic demonstration that water/hydroxide is a
ligand to the active-site metal in this family of molybdenum-containing enzymes (19, 20), this mechanistic possibility must be considered. Alternate mechanisms in which either the Mo=O or Mo-OH group
represents the catalytically labile oxygen have been considered (4) and are shown in Scheme 1. The initial
MoIVO(SH)(OR) intermediate of the catalytic sequence (21)
may be arrived at either by proton abstraction from C-8 of substrate (this site is quite acidic for a carbon acid, with a
pKa of ~14 (22)) followed by nucleophilic attack
on the electron-deficient MoVI=O unit (Scheme
1A) or by base-assisted nucleophilic attack on C-8 of
substrate by the metal-bound hydroxide (C-8 has also been shown to be
susceptible to nucleophilic attack (23)) followed by hydride transfer
from C-8 to the Mo=S group (Scheme 1B).
INTRODUCTION
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Abstract
Introduction
References
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Scheme 1.
Alternate reaction mechanisms for
xanthine oxidase.
In this work, we present evidence that the oxygen incorporated into
substrate in the course of the hydroxylation chemistry is derived from
the Mo-OH moiety of the molybdenum coordination sphere rather than
Mo=O, with the implication that the chemistry proceeds via the
mechanism that is shown in Scheme 1B.
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MATERIALS AND METHODS |
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Xanthine oxidase was isolated from unpasteurized cow's milk according to previously described methods (24). The enzyme was stored in liquid nitrogen in 0.1 M sodium pyrophosphate buffer, pH 8.5, containing 0.3 mM EDTA and 1.0 mM sodium salicylate until needed, the salicylate being removed by chromatography on Sephadex G-25 prior to use.
UV-visible absorption spectra and routine enzyme assays were performed with a Hewlett-Packard 8452 diode array spectrophotometer interfaced to a Hewlett-Packard Chemstation computer. Stopped-flow experiments were performed with a Kinetic Instruments stopped-flow apparatus equipped with an OLIS data collection system. Electron paramagnetic resonance spectra were obtained at 150 K with a Brüker ER 300 spectrometer equipped with an ER 035M NMR gauss meter and a Hewlett-Packard 5352B microwave frequency counter. Modulation frequency of 100 kHz, modulation amplitude of 2 G, sweep width of 400 G (from 3200 to 3600 G), and 10-milliwatt power were the standard instrument parameters for data acquisition. Simulations were performed using a software package developed by Dr. Graham George (Stanford Synchrotron Radiation Laboratory), running on a DEC Alpha workstation under OpenVMS.
Pre-steady-state kinetic studies of xanthine oxidase with 2,5-dihydroxybenzaldehyde and 2-aminopteridine-6-aldehyde were carried out under anaerobic conditions at 10 and 25 °C, respectively. The former reaction was monitored at 460 nm following the bleaching of the flavin and iron-sulfur centers that occurs upon reduction of the enzyme by substrate. The latter reaction was monitored at 620 nm following the absorbance increase associated with formation of the oxidized enzyme-substrate complex. These experiments were performed by making the enzyme in appropriate buffer anaerobic by repeated evacuation and flushing with pure argon for at least 1.5 h in a tonometer. Solutions of substrate were made anaerobic by dilution of fresh stocks (PTA dissolved in diluted KOH and 2,5-dihydroxybenzaldehyde in double-distilled H2O) into the appropriate buffer and bubbling the solutions with O2-free argon for at least 15 min.
Reversed-phase HPLC separation of 2,5-dihydroxybenzaldehyde and 2,5-dihydroxybenzoic acid was carried out using a Beckman HPLC system equipped with an ODS Hypersil 200 × 46-mm column (5-µm pore size; Hewlett-Packard), eluting with a degassed 70% methanol and 30% double-distilled water mixture as the mobile phase; the flow rate was 1 ml/min, and the eluate was monitored at 350 nm. The product of the enzyme reaction with 2,5-dihydroxybenzaldehyde was obtained by incubation of the enzyme with 2,5-dihydroxybenzaldehyde for 1.5 h, followed by centrifugation through a 50-kDa cutoff membrane to remove the enzyme and dilution with 70:30 (v/v) MeOH/H2O. Samples were analyzed by the HPLC system and compared with authentic 2,5-dihydroxybenzaldehyde and 2,5-dihydroxybenzoic acid to verify the product of the enzyme reaction.
The pKa values for 2,5-dihydroxybenzaldehyde and PTA
were independently determined spectrophotometrically. The protonated (neutral) form of 2,5-dihydroxybenzaldehyde exhibits a maximum absorbance at 358 nm, which shifts upon deprotonation to 410 nm. The
pKa was obtained by fitting the absorbance at 410 nm
as a function of pH to the equation A = (AHA·[H+] + AA·Ka)/([H+] + Ka), where AHA is the
absorbance of protonated substrate, AA
is the absorbance of the deprotonated form, and Ka
is the acid dissociation constant. The absorption spectrum of PTA is
also pH-dependent, exhibiting absorbance maxima at 280 and
370 nm at high pH, shifting upon deprotonation to 312 nm. The
pKa value for PTA was obtained as described above following this absorbance change.
Steady-state kinetic measurements with 2,5-dihydroxybenzaldehyde as
substrate were performed from pH 5.5 to 9.4 at 25 °C following the
absorbance increase at 320 nm for the conversion of the aldehyde to the
corresponding acid. Since both the substrate and product exhibit
pH-dependent spectra, it was necessary to independently determine the extinction change for oxidation at each pH. The
values at 320 nm ranged from 2.8 mM
1
cm
1 at pH 5.5 to 4.0 mM
1
cm
1 at pH 9.4. The concentration of functional enzyme
active sites ranged from 13 to 26 nM in these experiments,
and the substrate concentration ranged from 20 to 2000 µM, corresponding to a range at least between 0.3 and 3 times the Km values at each pH. The observed
catalytic velocities were fit to the standard hyperbolic equation
(Equation 1), where each term has its conventional definition.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
2-Aminopteridine-6-aldehyde, 2,5-dihydroxybenzaldehyde, and
2,5-dihydroxybenzoic acid were purchased from Aldrich; all other reagents were from Sigma. The following buffers were utilized in this
work: 0.1 M MES, pH 5.5-6.5; 0.1 M MOPS, pH
6.75-7.7; 0.1 M EPPS, pH 8.0-8.5; and 0.1 M
CHES, pH 9.0-9.4. Each buffer was supplemented with 0.3 mM
EDTA and 0.1 N KCl. All reagents were of the highest purity
commercially available. H217O (49% enriched)
was obtained from Cambridge Isotope Laboratories. Enzyme in
H217O was prepared by lyophilization of an
enzyme sample in 0.1 M pyrophosphate, pH 8.5, to dryness
and taking up in an appropriate volume of labeled water. Enzyme in
labeled solvent was used within 5 min of preparation to avoid
noncatalytic exchange of label into the molybdenum center of the
oxidized enzyme.
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RESULTS AND DISCUSSION |
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Steady-state Kinetics of Xanthine Oxidase with 2,5-Dihydroxybenzaldehyde-- As a preliminary to a steady-state kinetic analysis, the effectiveness of 2,5-dihydroxybenzaldehyde as substrate for xanthine oxidase was examined. 2,5-Dihydroxybenzaldehyde was found to be efficiently oxidized to 2,5-dihydroxybenzoic acid, as indicated by a UV-visible spectral comparison of the reaction product with authentic 2,5-dihydroxybenzoic acid. HPLC was also performed with the product obtained after reaction of the enzyme with 2,5-dihydroxybenzaldehyde for 1.5 h. The retention time of the product thus obtained (1.38 min) was the same as that of authentic 2,5-dihydroxybenzoic acid under the experimental conditions (see "Materials and Methods"). No substrate peak was seen after 1.5 h of reaction, indicating that xanthine oxidase quantitatively converts 2,5-dihydroxybenzaldehyde to 2,5-dihydroxybenzoic acid, as expected given the favorable driving force for the reaction.
Steady-state measurements of the xanthine oxidase-catalyzed oxidation of 2,5-dihydroxybenzaldehyde were carried out at 25 °C, monitoring product formation at 320 nm.2 By contrast with the behavior seen with xanthine, no obvious excess substrate inhibition was observed with 2,5-dihydroxybenzaldehyde even at the highest concentrations used (ranging from 2 mM at pH 9.4 to 560 µM at pH 7). The apparent kcat and Km values obtained by hyperbolic fits to the steady-state kinetic data are listed in Table I. Fig. 1 shows the pH dependence of the kinetic parameter (kcat/Km)app, which reflects the effective second-order rate constant for the reaction of free substrate with free enzyme in the low substrate concentration regime. These data describe a bell-shaped curve, which can be fitted using a double-ionization equation (see "Materials and Methods") to yield two pKa values that govern catalysis. The lower pKa of 6.3 ± 0.1 obtained from this analysis agrees well with that determined previously for an active-site residue essential in the hydroxylation of xanthine and lumazine (2,4-dihydroxypteridine) by xanthine oxidase (pKa ~ 6.5). The higher pKa of 8.6 ± 0.1 from this work agrees well with that of the substrate 2,5-dihydroxybenzaldehyde, which has been independently determined spectrophotometrically (pKa = 8.4) (Fig. 1, inset) and by acid-base titration (pKa 8.5) (data not shown). The implication is that it is the neutral rather than the ionized form of substrate that is acted upon by the enzyme. The overall pH dependence of the reaction is comparable to that of enzymatic action on both xanthine and lumazine (26), suggesting strongly that xanthine oxidase hydroxylates aromatic heterocycles and aldehydes by the same principal mechanism, one involving base-assisted catalysis on an unionized (neutral) substrate.
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Reductive Half-reaction Kinetics with 2,5-Dihydroxybenzaldehyde-- Studies of the reductive half-reaction of xanthine oxidase with 2,5-dihydroxybenzaldehyde were carried out at 10 °C in a stopped-flow apparatus following the loss of absorption at 460 nm due to reduction of the flavin and iron-sulfur centers of the enzyme under anaerobic conditions. It is known that full reduction of xanthine oxidase requires 3 eq of substrate and that this reaction goes essentially to completion in the case of xanthine. On the other hand, xanthine is unable to fully reduce xanthine dehydrogenase, owing to the establishment of unfavorable internal redox equilibria with this enzyme (27). To determine whether the reaction of the aldehyde substrate with the oxidase also goes to completion under the present experimental conditions, the total absorbance change elicited by a 3-fold stoichiometric excess of 2,5-dihydroxybenzaldehyde was compared with that induced by a comparable amount of xanthine when the enzyme concentration and other conditions were the same. It was found that on addition of 2,5-dihydroxybenzaldehyde under anaerobic conditions, the level of xanthine oxidase reduction was the same as that by xanthine (data not shown), indicating that xanthine oxidase is indeed completely reduced by 2,5-dihydroxybenzaldehyde.
The reaction of xanthine oxidase with a pseudo first-order excess of 2,5-dihydroxybenzaldehyde under anaerobic conditions was found to exhibit two well resolved kinetic phases. The rate constant for the faster phase of the reaction exhibits hyperbolic dependence on substrate concentration, yielding values for the limiting rate constant (klim) and substrate dissociation constant (Kd) that are pH-dependent. klim/Kd for this faster phase exhibits a bell-shaped pH dependence with a lower pKa of 6.5 ± 0.2 and a higher pKa of 8.3 ± 0.2 (Fig. 2), comparable to that seen in the steady-state experiments above and consistent with the reductive half-reaction being principally rate-limiting during turnover, as is the case for turnover with xanthine (25). The results again implicate an active-site residue that must be deprotonated for the reaction to occur.
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The above results are consistent with a reaction mechanism involving nucleophilic attack on the substrate carbonyl by a Mo-OH group, with base-assisted proton abstraction from the attacking hydroxyl by a residue in the active site of the enzyme (Scheme 1B). Recently, the crystal structure of aldehyde oxidase from Desulfovibrio gigas was solved at 1.8 Å, and on the basis of this structure, a mechanism of aldehyde hydroxylation was proposed that involves similar chemistry (19).3 Specifically, it was proposed that Glu-869 at the active site abstracts a proton from molybdenum-bound water/hydroxide and thereby facilitates nucleophilic attack on the substrate carbonyl to generate, after hydride transfer, a MoIV complex with product coordinated to the metal via the newly formed -OH. Xanthine oxidase and aldehyde oxidase share a highly homologous amino acid sequence in the vicinity of their molybdenum centers that includes this glutamate residue (VGE869LPL for the D. gigas protein versus VGE1261PPL for the bovine enzyme). We thus suggest that Glu-1261 of xanthine oxidase plays a role analogous to that proposed for Glu-869 of aldehyde oxidase, in which case the pKa associated with the enzyme is most likely to be associated with this residue. Although the observed pKa of ~6.4 is rather high for a glutamate, it is not unreasonable: the pKa for Glu-35 in lysozyme has been reported to be 6.5 in the absence of substrate and to shift to ~8.2 on binding glycol chitin (31). We note that it is unlikely that the ionization governing the acid limb of the klim/Kd pH profiles is due to the Mo-OH group itself, as x-ray absorption studies of xanthine oxidase have demonstrated quite unambiguously that at pH 8.5 (well above the pKa for the active-site residue), the molybdenum center possesses only a single Mo=O group rather than the two that would be expected were the Mo-OH to deprotonate (32).
The slower phase of the reaction of xanthine oxidase with
2,5-dihydroxybenzaldehyde, which accounts for only 10-20% of the total observed spectral change at high substrate concentrations, has
observed rate constants between 0.1 and 0.2 s1 that are
not strikingly dependent on substrate concentration. The rate constant
associated with the slower phase of the reaction is too large to
reflect the slow reduction of nonfunctional enzyme that is known to
occur with xanthine oxidase (25, 33), and it appears that the slower
phase most likely involves processes taking place during the reaction
of the enzyme with a second or (more likely) third eq of substrate
under the present conditions of pseudo first-order excess of substrate.
Such kinetic complexity has also been observed in the reaction of
xanthine with xanthine dehydrogenase (34). To test this idea, a
single-turnover experiment was performed in which the enzyme rather
than substrate was present in excess. When excess xanthine oxidase was
reacted with 2,5-dihydroxybenzaldehyde (5:1 xanthine
oxidase/substrate), only a single kinetic phase was observed,
corresponding in rate to the fast phase of the reaction seen with
excess substrate under comparable concentrations of substrate. This
indicates that the slower phase of the reaction seen under conditions
of excess substrate involves the reaction of the second or third eq of
substrate with the enzyme, possibly rate-limited by product release
from the molybdenum center in a prior catalytic sequence. As this
slower phase of the reaction does not reflect the intrinsic initial
reactivity of substrate with the molybdenum center of the enzyme and
given that it contributes relatively little to the overall absorbance
change seen in the course of the reaction, it has not been investigated
further here.
Reaction Kinetics with 2-Aminopteridine-6-aldehyde--
The above
work indicates that the reaction of xanthine oxidase with an aldehyde
substrate, through the first irreversible step of the reaction as
traced by kcat/Km and
klim/Kd, involves
base-assisted attack on neutral substrate. A series of reductive
half-reaction and steady-state studies with 2-hydroxy-6-methylpurine, xanthine, and lumazine as substrate have been examined previously, with
the evidence obtained indicating that the hydroxylation proceeds through two intermediates, corresponding to MoIV-product
and MoV-product complexes (21, 26). To investigate early
steps in the catalytic sequence seen with aldehyde substrates that are upstream from formation of the MoIV-product intermediate,
experiments following the reaction of xanthine oxidase with PTA have
been performed. PTA is a slow substrate for xanthine oxidase that is
hydroxylated to the corresponding carboxylic acid by the enzyme (35).
This substrate is interesting in that its reaction with the oxidized
enzyme gives rise to a transient long-wavelength absorbance (Fig.
3A) (35). At pH 8.5, the rate
constant associated with formation of this species exhibits hyperbolic
dependence on substrate concentration, indicating that formation of the
long wavelength-absorbing species involves two distinct kinetic
steps: Eox + S Eox·S
Eox·S*,
where Eox·S* represents the long
wavelength-absorbing oxidized enzyme-substrate complex.4 We
have examined the pH dependence of this reaction following (klim/Kd)app
obtained from hyperbolic fits to plots of the observed rate constant versus PTA concentration as a function of pH. In contrast to
the above work with 2,5-dihydroxybenzaldehyde as substrate, the plot of
(klim/Kd)app as a
function of pH is sigmoidal, indicating only a single
pKa with a value of 7.6 ± 0.2 (Fig.
3B). This is in fair agreement with the substrate
pKa of 7.2 ± 0.1, and we consider the observed
pKa to be that of the substrate. The implication, as
before, is that it is the neutral rather than the monoanionic form of
substrate that binds to the enzyme. The distinct difference in pH
profile for
(klim/Kd)app for
formation of Eox·S* in the
reaction with PTA (i.e. sigmoidal) as compared with that for
klim/Kd for the reductive
half-reaction with 2,5-dihydroxybenzaldehyde or xanthine
(i.e. bell-shaped) indicates that the catalytically essential active-site base is not required for formation of the long
wavelength-absorbing intermediate accumulating in the course of the
reaction with PTA. The implication is that the chemistry of
hydroxylation, which requires the active-site base, has not taken place
in forming the long wavelength-absorbing species, consistent with this
species being formulated as MoVI·S species rather than
MoIV·P. By way of confirming this conclusion, we find
that addition of pterin-6-carboxylic acid to dithionite-reduced
xanthine oxidase does not give rise to the observed long
wavelength-absorbance increase. There is in fact no discernible
spectral change observed on addition of pterin-6-carboxylic acid to the
reduced enzyme. In contrast, the reaction of xanthine oxidase with
lumazine gives rise to a transient absorbance in the 600-700 nm region
that has been shown to be due to the MoIV-violapterin
(2,4,7-trihydroxypteridine) complex, and the identical complex can be
conveniently generated by addition of the product violapterin to
dithionite-reduced enzyme under anaerobic conditions (26). Taken
together, these results demonstrate that the observed long-wavelength
absorbance increase seen with PTA is not due to a
MoIV-product complex, but is instead a
MoVI-substrate complex in which the chemistry leading to
substrate hydroxylation has not yet taken place.
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Binding of 2,5-Dihydroxybenzaldehyde and 2,5-Dihydroxybenzoic Acid to Oxidized and Reduced Xanthine Oxidase-- In previous work, it has been shown that a number of enzyme products and product analogs bind to reduced xanthine oxidase with reasonably high affinity (Kd ~ 18 µM) and elicit a small spectral change at the molybdenum center upon binding (37). To determine whether 2,5-dihydroxybenzoic acid interacts with the enzyme in the same way, titrations of both oxidized and reduced enzyme with 2,5-dihydroxybenzoic acid were performed. We find that 2,5-dihydroxybenzoic acid binds the oxidized enzyme and produces a spectral change similar to that of 8-bromoxanthine, a spectral change known to be associated with the enzyme flavin rather than its molybdenum center (37). The Kd for 2,5-dihydroxybenzoic acid binding is large, ~8 mM. Titration of dithionite-reduced xanthine oxidase with 2,5-dihydroxybenzoic acid and 2,5-dihydroxybenzaldehyde has also been attempted, but no obvious spectral change is observed (data not shown).
Xanthine oxidase frequently exhibits excess substrate inhibition (37, 38), a phenomenon thought to be associated with substrate binding to reduced rather than oxidized forms of the enzyme generated in the steady state (37, 38). In this work, no obvious excess substrate inhibition was observed with 2,5-dihydroxybenzaldehyde, although product inhibition by the corresponding carboxylic acid did exist. To investigate how product influences the enzyme kinetics, reductive half-reaction studies have been performed with varying concentrations of product (0, 100, 200, and 500 µM) mixed at a given concentration of substrate (50 or 150 µM), again following the reaction by the absorbance change at 460 nm. At pH 7, we find that the rate constants for both the faster and slower phases of the reaction decrease with increasing product concentration, although the total amplitude for the spectral change associated with the reaction does not change. These results are consistent with the above observation that enzyme reduction goes to completion and is not backed up even in the presence of excess product. To evaluate the inhibitory effect of the product on xanthine oxidase, steady-state experiments have been performed (again using air-equilibrated buffer). We find that the product is a competitive inhibitor with respect to the substrate, with a plot of apparent Km as a function of product concentration giving a value for Ki of 260 µM (data not shown). The competitive inhibition pattern observed for the product with respect to the substrate indicates that both substrate and product bind to the oxidized molybdenum center, which contrasts with the observation that urate and 8-bromoxanthine preferentially bind to the reduced molybdenum center. Although 2,5-dihydroxybenzoic acid also binds to the flavin center of the oxidized enzyme, it is rather weak (~8 mM) compared with its binding to the oxidized molybdenum center (~260 µM). It is to be emphasized that the inhibition seen with 2,5-dihydroxybenzaldehyde and its product is different from that for xanthine and urate in two aspects: no excess substrate inhibition is observed with the aldehyde substrate in initial velocity steady-state experiments, and product inhibition is competitive, the result of product binding to the oxidized enzyme rather than the reduced enzyme.
Identification of the Catalytically Labile Oxygen in the Active Site of Xanthine Oxidase-- To establish whether the catalytically labile oxygen of the active site of xanthine oxidase is the Mo=O or Mo-OH moiety known to be present in the molybdenum coordination sphere, we have investigated the exchange of 17O from labeled solvent into the molybdenum center in the course of a single turnover. After an initial turnover event, the site of the catalytically labile oxygen will be regenerated with oxygen derived from solvent, and the isotopically labeled oxygen from solvent can be used as a probe of the active site. This approach takes advantage of previous observations that after exhaustive exchange with solvent, several of the MoV EPR signals of xanthine oxidase exhibit magnetic coupling to one strongly coupled 17O nucleus and frequently a second much more weakly coupled one as well (17, 39-41). It has been demonstrated in suitable model compounds that Mo-OH groups couple strongly to the unpaired electron spin (aav ~ 6.5 G); Mo=O groups are much more weakly coupled (aav ~ 2.2 G) (17). EPR thus represents a uniquely appropriate tool whereby exchange into the Mo-OH and/or Mo=O site under single-turnover conditions can be examined on a catalytic time scale.
Fig. 4 (spectrum A) shows the EPR spectrum observed when 150 µM deflavoxanthine oxidase is reacted with 500 µM 1-methylxanthine in unlabeled 0.1 M pyrophosphate, pH 8.5, under aerobic conditions and frozen in dry ice/acetone after ~200 ms of reaction. The observed EPR signal is of the expected "rapid Type 1" form typically seen with this substrate, exhibiting a doublet-of-doublets on the low-field gz feature (due to the presence of two inequivalent protons in the signal-giving species (41)). Spectrum B shows the spectrum observed when the experiment is repeated in water that was 49% enriched in 17O. The spectrum is quite distinct from spectrum A, particularly in the gz region, where the spectral resolution is apparently poorer, and in the central portion of the spectrum, where the strong positive feature in the H216O sample is replaced by a considerably weaker doublet in H217O. Although hyperfine splitting due to the I =5/2 nucleus of 17O is not evident in spectrum B, the observed spectrum is essentially identical to that observed when the enzyme is exhaustively exchanged with H217O prior to generation of the EPR signal (40). This signal has been demonstrated in computer simulations to arise from a species possessing a single strongly coupled 17O nucleus, with aav ~ 7 G. The hyperfine structure arising from the nuclear spin of 17O is more evident when spectrum B is corrected for the 51% 16O present in the sample, as shown in spectrum C. The dashed line (spectrum D) shows a simulation of the spectrum using a1,2,3 = 16, 3, and 2 G (aav = 7 G), and the fit to the data is seen to be quite good. These values are in excellent agreement with simulations of spectra obtained in exhaustively labeled enzyme (17, 39-41) and are clearly in the range of splittings observed for Mo-OH groups in model compounds (17). In particular, the good fit to the high field features (~3450 G) indicates the presence of a strongly coupled oxygen atom.
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On the time scale of the present experiment, the reaction subsequent to the initial reduction of the enzyme by the first eq of substrate and incorporation of 17O into the active site has three possible fates. 1) A portion of the enzyme will not have time to react with a second eq of substrate and will be reduced only to the level of 2 electron eq. Under the present reaction conditions (0.1 M pyrophosphate buffer, pH 8.5), the electron distribution within this 2-electron-reduced enzyme is heavily in favor of reduction of the two iron-sulfur centers over that of the molybdenum (25), and as a result, this enzyme population will be EPR-silent at 150 K.5 2) A second portion of the enzyme, having been reduced to the level of 2 electron eq in the first turnover and possessing MoVI after transfer of the reducing equivalents obtained from substrate to the iron-sulfur centers, will have reacted with a second eq of substrate to give fully reduced, 4-electron deflavoxanthine oxidase; this enzyme population possesses MoIV and must be EPR-silent at 150 K.6 3) The remainder of the enzyme, having also been reduced to the level of 2 electron eq in the course of the first turnover, will also have bound substrate, but not yet have advanced into a second catalytic sequence. To the extent that this enzyme population possesses MoV or MoIV rather than MoVI, it will be indeed unable to react with substrate. The effect of substrate binding on the reduction potentials of the molybdenum center is in fact such that the MoV oxidation state is significantly stabilized by substrate binding to the active site of partially reduced enzyme (34, 39), a fact that accounts for the observed inhibition of the enzyme during turnover under conditions of high substrate concentration (5). In the present experiment, the net result is that ~25% of the functional enzyme in the reaction mixture is trapped after the first turnover in a MoV·S state that specifically gives rise to the rapid Type 1 EPR signal when 1-methylxanthine is used as substrate. The clear implication from the above experimental result is that after a single turnover, the oxygen atom is incorporated into the molybdenum center at a strongly coupled site (aav ~ 7 G) and, on the basis of previous work with model compounds (17), is unquestionably the Mo-OH group rather than Mo=O. The results are consistent with an overall sequence of events taking place in the enzyme active site as indicated in Scheme 2.
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It is known that reduction of the molybdenum center gives rise to much
more rapid noncatalytic exchange of solvent oxygen into the
molybdenum coordination sphere (40, 41), and it is conceivable that
under the present reaction conditions, the observed incorporation of
17O into the molybdenum center might be due to a rapid
noncatalytic exchange into the active site rather than to turnover
per se. To establish that this is not the case on the time
scale of the present experiment, unlabeled deflavoxanthine oxidase in
H217O was partially reduced by reaction with
sodium dithionite in an EPR tube in the presence of the substrate
analog 8-bromoxanthine, followed by freezing after ~5 s.
8-Bromoxanthine is not hydroxylated by xanthine oxidase, but does bind
to the active site of the partially reduced enzyme to elicit a "rapid
Type 2" MoV EPR signal, as reflected in a characteristic
1:2:1 triplet feature (arising from the strong coupling of two
equivalent protons) evident in the low-field gz
feature of the signal (37). The spectrum observed is shown in Fig. 4
(spectrum E) and is indistinguishable from that seen in
H216O; in particular, the 1:2:1 triplet of
gz is readily evident. This result indicates that no
detectable solvent exchange has occurred in the much longer time
interval between reduction of the enzyme and freezing than was used in
the experiment with 1-methylxanthine; the exchange observed in the
experiment with 1-methylxanthine must therefore be due to catalytic turnover.
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CONCLUSIONS |
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In this work, we have investigated the mechanism of aldehyde hydroxylation by xanthine oxidase by both steady-state and reductive half-reaction kinetics and examined the nature of the catalytically labile oxygen in the enzyme active site by EPR. On the basis of the similarity of the bell-shaped pH profiles for aldehyde and heterocycle hydroxylation as catalyzed by xanthine oxidase (this work and Ref. 26, respectively), it is extremely likely that the mechanism of hydroxylation of both aldehydes and heterocycles by xanthine oxidase is essentially the same, involving base-assisted nucleophilic attack on substrate. In light of the crystal structure of the related aldehyde oxidoreductase from D. gigas and the proposed role of Glu-869 of this enzyme in catalysis (19), we suggest that the homologous residue in xanthine oxidase, Glu-1261, represents the active-site base of xanthine oxidase. (Ultimately, proof of this will require development of a suitable expression system followed by site-directed mutagenesis of this residue.) The pH dependence of the reaction of enzyme with 2-aminopteridine-6-aldehyde indicates that the long wavelength-absorbing species formed at the completion of the reaction represents an Eox·S rather than an Ered·P complex.
The 17O experiments described here demonstrate that it is
the Mo-OH rather than the Mo=O group that is catalytically labile and
support a mechanism proceeding as indicated in Scheme 1B, in
which hydroxylation occurs via (base-assisted) nucleophilic attack of
an enzyme Mo-OH group on
substrate.7 Such a mechanism
is consistent with the structure of the molybdenum center of xanthine
oxidase as inferred from the crystal structure of the closely related
enzyme aldehyde oxidoreductase from D. gigas and has been
considered previously in light of the structural data (19). Similarly,
Wedd and co-workers (17) suggested earlier that a metal-coordinated
hydroxide rather than Mo=O might represent the catalytically labile
oxygen site of xanthine oxidase on the basis of EPR studies of model
compounds. Recently, Bray and co-workers (18) inferred from an ENDOR
study of the "very rapid" EPR signal that the Mo=O group does not
exchange with solvent in the course of catalysis (on the basis of their
inability to detect a second, weakly coupled 17O nucleus in
addition to the strongly coupled 17O of bound product) and
concluded that Mo-OH rather than Mo=O is the catalytically labile
oxygen site. The failure to detect a weakly coupled oxygen in this
experiment is not surprising, however, given the intrinsically weak
spectral signature of even a strongly coupled oxygen and the difficulty
of identifying a second, more weakly coupled nucleus in the presence of
a more strongly coupled one. Thus, the possibility cannot be excluded that a second oxygen site had indeed exchanged in the signal-giving species, but had escaped detection. The present work, unambiguously demonstrating that oxygen exchanges into a strongly coupled site of the
enzyme in the course of a single turnover, provides concrete and positive support of the Mo-OH group being the catalytically labile
oxygen site in the molybdenum center of xanthine oxidase.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AR 38917 (to R. H.).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 Medical
Biochemistry, 333 Hamilton Hall, 1645 Neil Ave., Ohio State University, Columbus, OH 43210-1218. Tel.: 614-292-3545; Fax: 614-292-4118; E-mail:
Hille.1{at}osu.edu.
The abbreviations used are: PTA, 2-aminopteridine-6-aldehyde; G, gauss; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; EPSS, 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid.
2 In these experiments, the buffer was air-equilibrated with an O2 concentration of ~240 µM at each pH. We note that the Kd for O2 is not very pH-dependent and that the Kd changes by only a factor of 2 over the pH range (25). Such a small difference in Kd as a function of pH should not affect much of the kinetics observed. In theory, for a two-substrate reaction with a ping-pong mechanism, when one substrate concentration is fixed, kcat/Km should not be affected, although the apparent kcat and Km values for the other substrate are different from the true kcat and Km values. In fact, there is good agreement on pH dependence between steady-state and reductive half-reaction results (see below).
3 The coordination geometry inferred from the crystallographic work is that of a distorted square pyramid, with an apical Mo=S group and the two sulfurs of the pterin cofactor, a Mo=O group, and a fifth oxygen ligand defining the basal plane (19, 20). This last ligand has been formulated as a water molecule on the basis of a Mo-O distance of ~2.2 Å, which is in the range for a metal-bound water (as opposed to hydroxide). As has been pointed out recently, however, this distance must be regarded as an upper limit for the true Mo-O distance, owing to series truncation artifacts around the heavy metal ion that occur in generation of the electron density map for the protein; these artifacts may in some cases lead to an overestimation of the true Mo-O distance by as much as 0.3 Å (28). It remains to be seen whether this is a concern in the case of the molybdenum center of the aldehyde oxidoreductase. A hydroxide ligand is preferred here on the following additional grounds. 1) In small inorganic complexes of MoVI, oxygen ligands are most frequently found as fully deprotonated Mo=O, occasionally as singly protonated Mo-OH, but only extremely rarely as fully protonated Mo-OH2 (29). 2) Computational studies have shown that Mo-OH is expected to be considerably more stable from a thermodynamic standpoint than Mo-H2O in the specific coordination geometry found in the active site of the aldehyde oxidoreductase (30).
4
The observed rate constant
(kobs) for this two-step mechanism can be solved
analytically (36) as follows: kobs = (k2·[S]/([S] + Kd)) + k2. A linear double-reciprocal plot is observed in the present case, indicating that
k
2 is very small and that step 2 is irreversible.
5 It is to be noted, however, that to the extent that a MoV signal is observed, it must arise from enzyme that has turned over once (and only once) and thus will possess 17O at the catalytically labile oxygen site. Also, although the reduced iron-sulfur centers are reduced and paramagnetic under the present experimental conditions, these EPR signals are too broad to be detected at liquid nitrogen temperatures.
6 Deflavoxanthine oxidase was used in the present experiment to avoid potential complications associated with multiple turnover with excess substrate under the present anaerobic conditions. By removing the FAD, the enzyme can react with no more than 2 eq of substrate, and the MoIV that results is EPR-silent. This ensures that all the EPR-active molybdenum can have reacted only once with substrate. It has been demonstrated that removal of the flavin does not affect the kinetic behavior of the molybdenum center (25).
7 It is to be noted that the Mo=O and Mo-OH sites are quite distinct and are not expected to become equivalent in the enzyme in the course of catalysis. The crystal structure of the D. gigas aldehyde oxidase demonstrates quite clear differences in Mo-O bond lengths and protein environments for the two ligands. In addition, it should be emphasized that if the Mo=O and Mo-OH (as referred to here) ligands become equivalent in the course of the catalytic cycle, a 50% washout of the label that is subsequently incorporated into product under single-turnover conditions is to be expected; such washout is not observed experimentally.
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REFERENCES |
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