Kinetic model of oxidation catalyzed by xanthine oxidase - The final enzyme in degradation of purine nucleosides and nucleotides

Article abstract of DOI:10.1081/NCN-200060006

A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a broad pH range (5.8-9.0) are best described by an equation which is a rational function of degree 2:3 and 2:2, respectively. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200060006

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KINETIC MODEL OF OXIDATION CATALYZED BY XANTHINE
OXIDASE—THE FINAL ENZYME IN DEGRADATION OF
PURINE NUCLEOSIDES AND NUCLEOTIDES
a
a
b c
a
Kinga Banach , Elżbieta Bojarska , Zygmunt Kazimierczuk
, Lucyna Magnowska &
a
Agnieszka Bzowska
a
Department of Biophysics , Institute of Experimental Physics, University of Warsaw ,
Warsaw, Poland
b
Institute of Chemistry, Agricultural University , Warsaw, Poland
c
Laboratory of Experimental Pharmacology , Polish Academy of Sciences Medical Research
Center , Warsaw, Poland
Published online: 15 Nov 2011.
To cite this article: Kinga Banach , Elżbieta Bojarska , Zygmunt Kazimierczuk , Lucyna Magnowska & Agnieszka Bzowska
2005) KINETIC MODEL OF OXIDATION CATALYZED BY XANTHINE OXIDASE—THE FINAL ENZYME IN DEGRADATION OF PURINE
(
NUCLEOSIDES AND NUCLEOTIDES, Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 465-469, DOI: 10.1081/NCN-200060006
To link to this article: http://dx.doi.org/10.1081/NCN-200060006
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):465–469, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060006
KINETIC MODEL OF OXIDATION CATALYZED BY XANTHINE
OXIDASE—THE FINAL ENZYME IN DEGRADATION OF PURINE
NUCLEOSIDES AND NUCLEOTIDES
5
Kinga Banach and El ˙z bieta Bojarska
Department of Biophysics, Institute of
Experimental Physics, University of Warsaw, Warsaw, Poland
5
Zygmunt Kazimierczuk
Institute of Chemistry, Agricultural University, Warsaw,
Poland and Laboratory of Experimental Pharmacology, Polish Academy of Sciences Medical
Research Center, Warsaw, Poland
5
Lucyna Magnowska and Agnieszka Bzowska
Department of Biophysics, Institute
of Experimental Physics, University of Warsaw, Warsaw, Poland
5
A new kinetic model is presented for analysis of experimental data of oxidation process catalyzed by
milk xanthine oxidase. The kinetics for two substrates, xanthine and its analog 2-chloroadenine, in a
broad pH range (5.8---9.0) are best described by an equation which is a rational function of degree 2:3
and 2:2, respectively.
Keywords Xanthine Oxidase, Enzyme Kinetics, Purine Degradation, Biological Oxidation,
Non-Michaelis–Menten Kinetics
INTRODUCTION
Xanthine oxidase (XO) is a molybdeno-flavoprotein complex involved in the
final stage of degradation of nucleic acids’ components. It catalyzes the oxidation of
hypoxanthine (Hx) to xanthine (Xan) and subsequently to uric acid that is the end
[1]
product of purine catabolism in man. Accumulation of uric acid in human blood
plasma (hyperuricemia) leads to formation and deposition of sodium urate
[2]
microcrystals in joints, which results in gout. Allopurinol, an analog of Hx and a
weak XO inhibitor developed 30 years ago, is the only clinically used drug for the
[3]
treatment of hyperuricemia associated with chronic gout.
Supported by the Foundation for Development Diagnostic and Therapy (Warsaw, Poland) and by BST 833/
BF project from University of Warsaw.
Address correspondence to Agnieszka Bzowska, Department of Biophysics, Institute of Experimental Physics,
University of Warsaw, 93 Zwirki & Wigury, Warsaw 02-089, Poland; E-mail: abzowska@biogeo.uw.edu.pl
465
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466
K. Banach et al.
XO seems to play an important role that is not limited to oxidation pathway in
purine metabolism. It was suggested that the enzyme is involved in pathogenesis of
post-ischemic reperfusion tissue injury and in protection against bacterial infection,
toxic plants and metabolic xenobiotics by producing reactive oxygen species
[
4,5]
during the redox cycle.
Serum XO is significantly elevated in various path-
ological states like hepatitis, inflammation, malignancies and ageing, and it is
commonly accepted that reactive oxygen species generated by the enzyme are
involved in oxidative damage. Thus, the inhibition of this enzymatic pathway could
be beneficial in a broad spectrum of pathologies, and not only in treatment of
[
6]
gout.
XO-catalyzed oxidation of biological substrates (Hx and Xan), and inhibition of
the enzyme by compounds with potential medical applications, have been de-
scribed in the literature using the Michaelis–Menten equation. However, as
[
7]
outlined by Bardsley et al., experimental data obtained even for narrow ranges of
substrate concentration showed pronounced deviation from the hyperbolic kinetics.
Hence, interpretation of experimental data from XO-catalyzed processes in terms of
classical parameters (K , Vmax for substrates, and K for inhibitors) is inappropriate.
m
i
Another important problem that has been overlooked in the studies of XO is
the possible role of ionic forms of purine substrate in interactions with the enzyme
and in catalysis. Xan has a pK of 7.7, which is attributed to ionization of N-3
a
position. Hence, in many physiological and pathological conditions there is a
[8,9]
mixture of anionic and neutral forms of the substrate in solution.
This should be
taken into account when analyzing kinetic data and constructing kinetic and
molecular models of the XO catalytic mechanism.
EXPERIMENTAL
Xanthine oxidase from bovine milk (EC 1.2.3.2) and xanthine were purchased
from Sigma (St. Louis, MO). 2-chloroadenine (2ClAde) was synthesized according
[10]
the procedure described previously. Concentrations of the enzyme and substrates
in solutions were determined from absorbance data using the respective molar
ꢀ1
ꢀ1
extinction coefficients. For XO emax(280 nm) = 202 600 M cm was determined
based on the amino acid sequence P80457 obtained from SwissProt database.
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
For Xan emax(267 nm) = 10,300 M cm at pH 6.0, emax(270 nm) = 9,100 M cm
ꢀ
1
ꢀ1
at pH 7.5, e (276 nm) = 9,300 M cm at pH 9.0; for 2ClAde emax(264 nm)
max
ꢀ
1
ꢀ1
ꢀ1
ꢀ1
=
12,000 M cm
at pH 6.0, e (264 nm) = 11,900 M cm
at pH 7.5,
max
ꢀ
1
ꢀ1
emax(266 nm) = 11,100 M cm at pH 9.0. Due to low solubility, 2ClAde samples
were first dissolved in a slightly alkaline medium (0.01 NaOH) and then diluted with a
phosphate buffer of the appropriate pH value.
Enzymatic reactions have been performed at 25°C in 50 mM phosphate buffer
in the pH range 5.8–9.0. Ultraviolet absorption spectra were recorded with an
Uvikon 940 UV-VIS spectrophotometer (Kontron, Vienna, Austria), fitted with
thermostatically controlled cell compartments. Oxidation of Xan was monitored

K. Banach et al.
467
at 300 nm corresponding to the uric acid absorption. Formation of 8-hydroxy-2-
chloroadenine, the oxidation product of 2ClAde, was monitored at 285 nm. Initial
velocity of both processes was calculated using linear regression of absorbance
vs. time.
Nonlinear regression analysis of the experimental data was performed using
GraphPad Prism 3.0 (GraphPad Software Inc. ) and Origin 6.0 (Microcal) computer
software packages. Rational functions of degree 2:2, 2:3 (Eqs. 1 and2), 3:2 and 3:3
[
7]
were fitted to kinetic data of initial velocity (v ) vs. initial substrate concentration
o
(
co). Statistically significant decrease in the sum of residuals (as estimated with the
Snedecor’s F test at a 95% confidence level), residual plots, and P value from the
runs test were used to identify major deviations from the equation selected, and to
discriminate between various models.
FIGURE 1 Kinetic of oxidation of Xan (left panels) and 2CldAde (right panels) catalyzed by XO. Reactions
were conducted at 25°C in 50 mM phosphate buffer at pH values indicated. Fitting of the rational function of
degree 2:2 (Eq. 1) and of degree 2:3 (Eq. 2) to the initial velocity data v
o o
(c ) led to solid and broken lines,
respectively.

468
K. Banach et al.
RESULTS AND DISCUSSION
Our aim was to develop a suitable kinetic model for mammalian XO-mediated
catalysis by studying the oxidation process of the natural substrate Xan and its
structural analog 2ClAde over a wide range of the initial substrate concentrations
(
0.5–500 mM) and in a broad range of pH values (5.8–9.0). The pK = 7.7 of Xan
a
deprotonation indicates that both in our experiments and in biological conditions
there is a mixture of the neutral and monoanionic forms of Xan. The situation is
quite different for 2ClAde. The pK of 8.5 for this compound, which value was
a
determined by spectrophotometrical titration, indicates that 2ClAde exists
predominantly in the neutral form in the majority of biological environments.
The initial velocity data v (c ) obtained for both compounds at pH 6.0, 7.5, 9.0
o
o
are presented in Figure 1. (Data obtained at pH 5.8, 6.5, 7.0, 8.0 and 8.5 are not
shown). Kinetics at all pH values studied is clearly non-hyperbolic, hence the use of
the Michaelis–Menten equation is not adequate. The data could be described as
an apparent substrate inhibition, since maximal velocity is obtained for a relatively
low initial substrate concentration (20–80 mM, depending on pH and substrate
employed), and the reaction rates are lower for higher c . The rates of oxidation are
o
pH dependent. The maximum velocity is about 0.3, 0.6, and 0.6 U/mg for Xan, and
0.1, 0.2, and 0.2 U/mg for 2ClAde for pH 6.0, 7.5, and 9.0, respectively. Hence, pKa
of the substrate appeared to not influence the pH dependence of the maximal rate
of oxidation. However, substrate concentration corresponding to the maximal rate
is pH dependent for Xan, and pH independent for 2ClAde (Figure 1), suggesting
that the neutral form of Xan binds stronger to XO than the monoanion.
Nonlinear regression analysis of the kinetic data yielded adequate fits for
rational functions of degree 2:2 for 2ClAde (Eq. 1) and 2:3 for Xan (Eq. 2, see
Figure 1):
2
o
jc þ ic
o
v ðc Þ ¼
ð1Þ
ð2Þ
o
o
2
o
1
þ mc þ lc
o
2
o
2
o
jc þ ic
o
v ðc Þ ¼
o
o
3
o
1
þ mc þ lc þ kc
o
These are the mathematical expressions describing several reaction mecha-
nisms; constants i, j, k, l, and m represents combination of rate constants and their
interpretation depends on the molecular mechanism.
Hence, in general, a rational function of degree at least 2:2 is necessary to
describe kinetic data for XO. Such a function describes several possible molecular
mechanisms of enzymatic reactions, e.g., allosteric interactions between enzyme
subunits, steady-state random systems and many others (e.g., Refs. [7,11,12]).
Steady-state kinetic data alone does not permit to distinguish unequivocally
between various mechanisms. However, it is clear from the data presented here that

K. Banach et al.
469
interpretation of kinetic data for XO on the basis of Michaelis–Menten model is
incorrect. This should be given special consideration when describing new
inhibitors of XO with potential medical applications. Determination of classical
inhibition types and inhibition constants (K ) is not feasible for XO inhibition and
i
may lead to incorrect conclusions regarding possible clinical application of
candidate compounds. In this case, IC50 values seem to be more adequate to
characterize inhibitory potency of new and previously synthesized analogues.
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