(Received for publication, February 14, 1994; and in revised form, December 16, 1994)
From the
The mechanism of inhibition of milk xanthine oxidase and
xanthine dehydrogenase by the tight binding inhibitor,
sodium-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5-triazine-4-olate
monohydrate (BOF4272), was studied after separation of the two isomers.
The steady state kinetics showed that the inhibition by these compounds
was a mixed type. One of the isomers had a K value of 1.2
10
M and a K
` value of 9
10
M, while the other isomer had a K
value of 3
10
M and a K
` value of 9
10
M. Spectral changes were not
observed by mixing either the oxidized or reduced form of the enzyme
with BOF-4272. The stopped-flow study and the effects of BOF-4272 on
various substrates showed that BOF-4272 bound to the xanthine binding
site of the enzyme. K
values of the
enzyme and one of the isomers, which has a higher affinity for the
enzyme, were also found to be 2
10
M for the active form of the enzyme and 7
10
M for the desulfo-form using fluorometric titration, and
the binding has stoichiometry of 1:1. The inhibitor could not bind to
the enzyme when the enzyme was previously treated with oxipurinol.
Xanthine dehydrogenase (EC 1.1.1.204) catalyzes the oxidation of
hypoxanthine to xanthine and of xanthine to uric acid with concomitant
reduction of NAD to NADH(1) . The enzyme is a dimer of
identical subunits with a M of about 150,000
containing four oxidation and reduction centers per subunit: one
molybdopterin, two iron sulfur centers, and one FAD. Mammalian xanthine
dehydrogenases exist as the NAD-dependent type in freshly prepared
samples(2, 3) , i.e. they exhibit high
xanthine-NAD reductase activity even in the presence of
O
(2, 3, 4, 5) . However,
during purification, most of the enzymes are converted to the
O
-dependent xanthine oxidase
(XO)(
)(2, 3, 6, 7) , i.e. they exhibit low xanthine-NAD reductase activity but high
xanthine-O
reductase activity even in the presence of
NAD(2, 3, 4, 5) . Xanthine interacts
with XO or xanthine dehydrogenase at the xanthine binding site, which
contains molybdopterin, the structure of which has been proposed and
recently reviewed(8, 9) . Two electrons are
transferred from xanthine to Mo(VI), reducing the metal to Mo(IV) while
another substrate, NAD or oxygen, reacts with FAD; the catalytic cycle
thus necessarily entails intramolecular electron transfer.
Xanthine-oxidizing enzymes are inactivated by cyanide in a reaction that removes an essential sulfur atom from the molybdenum to give the nonfunctional desulfo-form of the enzyme(10) . Desulfoxanthine oxidase occurs naturally and is present in all standard preparations but can be removed by affinity chromatography(11, 12) . Evidence for the existence of the desulfo-form was provided by Massey et al. (13) with the study of the interaction of the enzyme and the inhibitor, allopurinol. The sulfur atom necessary for catalytic activity has been shown by x-ray absorption spectroscopy to be present as a Mo=S group(14, 15, 16, 17, 18) . It has been shown that Mo=O and Mo=S groups dominate the coordination sphere in oxidized enzyme, with the remainder of the ligand positions occupied by thiolate or thioether. On reduction, the Mo=S group becomes protonated to form Mo-SH and is considered to be the group that accepts the C-8 proton from xanthine in the course of its conversion to uric acid(18) . However, the interaction of the substrate with the molybdenum center and the chemical steps involved in its conversion to product are poorly understood(9) .
The interaction of xanthine oxidase with an
inhibitor is of interest not only from the aspect of the mechanism of
enzyme reaction, but also from the medical aspects. First, as the
enzyme is involved in the final steps of uric acid production, an
inhibitor of xanthine oxidase is useful for remedy of hyperuricemia or
gout(19) . Second, as the O-dependent type of the
enzyme produces hydrogen peroxide and superoxide
(O
), the conversion from the
NAD-dependent type to the O
-dependent type is
hypothetically proposed to be responsible for causing post-ischemic
reperfusion injury(20) . Therefore, it is valuable to examine
whether prior treatment of tissue with an inhibitor is effective in
attenuating post-ischemic tissue injury. Allopurinol, a
pyrazolopyrimidine derivative, is a potent inhibitor of xanthine
oxidase and has been used for effective remedy for hyperuricemia or
gout. It has been reported that treatment of tissue with allopurinol is
effective in attenuating ischemic tissue
injury(21, 22, 23) . The mechanism of
inhibition of xanthine oxidase by allopurinol was studied in detail,
indicating that oxipurinol, a product from allopurinol by xanthine
oxidase, bound stoichiometrically to the reduced molybdenum
(IV)(13) . Although the inhibitor binds very tightly to the
enzyme, the inhibition is time dependent(13, 24) ,
and, therefore, it takes some time to inhibit the enzyme completely.
Furthermore, it is necessary to maintain an effective concentration of
the inhibitor to the organ because the inhibitor dissociates from the
enzyme by spontaneous oxidation of molybdenum to Mo(VI), with a
half-life of 300 min at 25 °C, with concomitant recovery of enzyme
activity(13) . However, in the experiments of post-ischemic
reperfusion injury, the maintenance of an effective concentration of
the inhibitor makes it difficult to discriminate whether the inhibitor
is effective due to the inhibition of xanthine oxidase or due to the
scavenging of O
because allopurinol has
been reported to be a direct scavenger of superoxide
anion(25) . Thus, the involvement of the enzyme in reperfusion
injury is still controversial.
Many other inhibitors of xanthine oxidase have been reported since allopurinol has been introduced(26) . However, none of them except allopurinol has been used for remedy of hyperuricemia or for experimental studies, probably because of less effectiveness than allopurinol or because of possible side effects of the inhibitor on animals or humans. Recently, a new potent inhibitor of xanthine oxidase has been introduced and found to be an effective inhibitor even in vivo. Its inhibitory effects on liver xanthine oxidase in vivo have been found to last longer than allopurinol (27) . However, the mechanism of inhibition and the strength of binding of the inhibitor have not been reported.
Here, we report the inhibitory effects of this compound on xanthine oxidase in vitro and the mechanism of inhibition of this compound.
Milk XO was purified by the methods of Ball(28) . The
active form of XO was separated from the inactive desulfo-form
according to the method of Nishino et al.(11) . The
prepared enzyme was more than 90% active, i.e. it exhibited an
activity to flavin ratio (AFR) of more than 190 (fully active enzyme
has a value of 210)(10, 13) . The desulfo-enzyme
(inactive form) was prepared by incubating the enzyme in 10 mM KCN for 2 h at 25 °C followed by gel filtration to remove
KCN(10) . Milk xanthine dehydrogenase was purified by the
method of Nakamura and Yamazaki(29) . Before use, xanthine
dehydrogenase was previously incubated with 10 mM dithiothreitol and was passed through Sephadex G-25 to remove
dithiothreitol. The dehydrogenase/oxidase activity ratio of xanthine
dehydrogenase as defined by Waud and Rajagopalan (6) was around
9.
Sodium-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1,5-a]-1,3,5-triazine-4-olate
monohydrate (BOF-4272) was a generous gift from the Otsuka
Pharmaceutical Factory Inc. (Naruto, Japan). This material contained
isomers as shown in Fig. 1. The method of synthesis of BOF-4272
and the chemical nature will be described elsewhere. ()The
isomers were separated by HPLC using a Chiralcel OD column (Daisel
Chemical Co., Tokyo) and a solution of hexane, ethanol, and formate
(700:300:4) as a solvent. In this chromatography, the(-)-isomer
was eluted at an earlier retention time than the other (+)-isomer,
which was confirmed with the authentic synthesized non-racemic
compounds, which were provided by the Ohtsuka Pharmaceutical Factory
Inc. The separated compounds were used for experiments. The
concentration of BOF-4272 was checked by determination of the
absorbance at 320 nm using the molar absorption coefficient of 23,500.
Figure 1: Structure of BOF-4272. Leftpanel,(-)-isomer; rightpanel, (+)-isomer.
Absorption spectra were recorded with an Hitachi U-3200 spectrophotometer.
Fluorescent spectra were recorded with an Hitachi 650-60 fluorescence spectrophotometer. Stopped-flow experiments were performed with an Applied Photophysics apparatus.
Activities with 0.15 mM xanthine as an electron donor and various artificial substrates as electron acceptors were determined spectrophotometrically by following the reduction of the substrate as follows: NAD (0.5 mM) at 340 nm, methylene blue (0.02 mM) at 665 nm, potassium ferricyanide (0.4 mM) at 420 nm, DCPIP (0.05 mM) at 600 nm, or cytochrome c (0.025 mM) at 550 nm. All these reactions were performed in 50 mM potassium phosphate buffer (pH 7.8) under aerobic conditions except for methylene blue. Reaction with methylene blue was performed under anaerobic conditions. Activities were also measured in the presence of 0.05 mM NADH as an electron donor under the same conditions.
where [EI], [E], and [I] represent the concentrations of the enzyme-BOF-4272 complex, free enzyme, and free BOF-4272, respectively.
Fluorescence intensity (Fluor) is due to both EI and I and is given by the equation
and by the equation for concentration of BOF-4272,
where k and k
represent the fluorescence intensity coefficient due to EI and I, respectively, and [I]
represents the concentration of BOF-4272 added to the mixtures.
Rearrangement of and brings about :
The value of k was obtained from
the initial slope of titration curves at lower concentrations of
inhibitor in the presence of XO and k
from the
slope of the plot of Fluorversus BOF-4272 in the
absence of enzyme. [EI]
, i.e. at any concentration of added BOF-4272, was calculated by
measuring fluorescence, [I]
and
[E]
were obtained by differences, and K
was calculated by substitution into the standard ().
Figure 2:
Time course of xanthine-O
activity of xanthine oxidase in the presence of BOF-4272 or
allopurinol. The reactions were followed at 295 nm in 3 ml of solution
containing 0.15 mM xanthine, 0.1 M pyrophosphate
buffer (pH 8.5), 0.2 mM EDTA, and 1 nM xanthine
oxidase (AFR = 193) in the absence of inhibitor (
), in the
presence of 3.3 µM allopurinol (
), or in the
presence of 0.033 µM BOF-4272(-)-isomer (
)
at 25 °C.
Figure 3:
Lineweaver-Burk plots of inhibition of
xanthine-O activity of xanthine oxidase in the presence of
BOF-4272(-)-isomer.
, without BOF-4272;
, 4
nM;
, 8 nM;
, 12 nM;
, 16
nM;
, 20 nM. Inset, the K
and K
`
values were obtained from secondary plots of the slopes of the
Lineweaver-Burk plots (
) and the apparent 1/V
(
) versus the inhibitor concentration,
respectively.
Figure 4:
Lineweaver-Burk plots of inhibition of
xanthine-phenazine methosulfate activity of xanthine oxidase with
BOF-4272(-)-isomer. , without BOF-4272;
, 5
nM;
,10 nM;
, 15 nM. Inset, secondary plot of apparent K
versus the inhibitor
concentration.
Figure 5: Time course of the reduction of milk xanthine dehydrogenase with xanthine or NADH in the presence of BOF-4272 (-)-isomer. Milk xanthine dehydrogenase (13 µM, dehydrogenase/oxidase activity ratio = 9, AFR = 120) in 0.1 M pyrophosphate buffer (pH 8.5) containing 26 µM BOF-4272(-)-isomer was mixed with an equal volume of the same buffer solution containing (1) 200 µM xanthine or (2) 100 µM NADH using a stopped-flow apparatus at 25 °C under anaerobic conditions, and absorbance of 450 nm was followed.
Figure 6:
Fluorometric titration of milk xanthine
oxidase with the inhibitor. A, 1 ml of active form of milk
xanthine oxidase (AFR = 200) was mixed with various volumes
(0200 µl) of 5.2 µM BOF-4272(-)-isomer in
0.1 M pyrophosphate buffer (pH 8.5) at room temperature.
Fluorescence (excitation at 320 nm; emission at 412 nm) was followed.
Dilution factors were recalculated. Original concentrations of xanthine
oxidase were as follows:
, without xanthine oxidase;
, 0.2
µM;
, 0.4 µM. B, titration of
the desulfo-form of milk xanthine oxidase. The desulfo-form of milk
xanthine oxidase (AFR = 3) was titrated with
BOF-4272(-)-isomer. Experimental conditions were the same as
above.
, without xanthine oxidase;
, 2 µM;
, 3 µM;
, 4 µM. C,
titration of oxipurinol bound active form xanthine oxidase with
BOF-4272(-)-isomer. Oxipurinol bound milk xanthine oxidase (AFR
= 200), which was previously mixed with 20 µM allopurinol, was titrated with BOF-4272(-)-isomer.
Experimental conditions were same as above.
, without xanthine
oxidase (control);
, 2 µM free xanthine oxidase;
, 2 µM oxipurinol bound xanthine
oxidase.
Figure 7: Separation of BOF-4272 liberated from xanthine oxidase by a chiralcel HPLC. BOF-4272 liberated from the enzyme by heat treatment was subjected to the reverse phase column followed by a Chiralcel OD column as described under ``Materials and Methods.'' Upperpanel, elution profile of racemic compound of BOF-4272 (control) using a Chiralcel OD column; middlepanel, BOF-4272 that bound to the active form of xanthine oxidase; lowerpanel, BOF-4272 that bound to the desulfo-form of the enzyme.
The results presented here show that BOF-4272 is a potent inhibitor of xanthine oxidase. All the data are consistent with the conclusion that BOF-4272 binds to the xanthine binding site, not to the NADH binding site. As xanthine or oxipurinol interacts with the molybdenum, it was strongly suggested that the inhibitor binds in close proximity to the molybdenum. However, due to the lack of spectral change upon incubation of BOF-4272 with either the reduced or oxidized form of enzyme and due to the fact that BOF-4272 binds tightly to both active and desulfo-forms of enzyme, it is likely that no atoms of BOF-4272 are in the coordination sphere of the molybdenum. Rather, steric factors between the inhibitor and the enzyme protein around the molybdenum seem to be important for tight binding. As the enzyme has been recently crystallized and the x-ray analysis of the crystal is now in progress (33) , it will also be interesting to crystallize the enzyme-inhibitor complex for analysis of the interaction between the inhibitor and the molybdenum site.
As
BOF-4272 binds tightly to both oxidized and reduced forms of the
enzyme, it can be expected to inhibit the enzyme for longer periods
than allopurinol in vivo, since the oxipurinol-inhibited
enzyme can be reactivated by spontaneous reoxidation of the Mo(IV) to
the Mo(VI) state. Actual in vivo studies showed that BOF-4272
inhibited rat liver xanthine dehydrogenase very effectively for a
longer period than allopurinol(27) . The advantages of using
this compound for experimental studies are that the inhibitory effect
of the compound is not time dependent, and it can be used at relatively
low concentrations because of its strong inhibitory effects on xanthine
dehydrogenase or XO. In addition, the compound is not a strong
scavenger of superoxide radicals. ()Furthermore, since this
compound has a (+)-isomer, which is a much weaker inhibitor than
the other(-), it may be used as a valuable control reagent for
study of the role of xanthine-oxidizing enzymes in pathogenesis. Such
usage of these compounds for experimental studies was recently
reported(34) .