Xanthosineand xanthine
Substrate propertieswithpurinenucleosidephosphorylases,and relevance
to otherenzyme systems
Gerasim Stoychev
1
, Borys Kierdaszuk
1
and David Shugar
1,2
1
Department of Biophysics, Institute of Experimental Physics, University of Warsaw, Poland;
2
Institute of Biochemistry and
Biophysics, Polish Academy of Sciences, Warsaw, Poland
Substrate properties of xanthine (Xan) and xanthosine
(Xao) for purinenucleoside phosphorylases (PNP) of
mammalian origin have been reported previously, but only
at a single arbitrarily selected pH andwith no kinetic con-
stants. Additionally, studies have not taken into account the
fact that, at physiological pH, Xao (pK
a
¼ 5.7) is a mono-
anion, while Xan (pK
a
¼ 7.7) is an equilibrium mixture of
the neutral and monoanionic forms. Furthermore the
monoanionic forms, unlike those of guanosine (Guo) and
inosine (Ino), and guanine (Gua) and hypoxanthine (Hx),
are still 6-oxopurines. The optimum pH for PNP from
human erythrocytes and calf spleen with both Xao and Xan
is in the range 5–6, whereas those with Guo and Gua, and
Ino and Hx, are in the range 7–8. The pH-dependence of
substrate properties of Xao and Xan points to both neutral
and anionic forms as substrates, with a marked preference
for the neutral species. Both neutral and anionic forms of
6-thioxanthine (pK
a
¼ 6.5 ± 0.1), but not of 2-thioxan-
thine (pK
a
¼ 5.9 ± 0.1), are weaker substrates. Phosphor-
olysis of Xao to Xan by calf spleen PNP at pH 5.7 levels off
at 83% conversion, due to equilibrium with the reverse
synthetic pathway (equilibrium constant 0.05), and not by
product inhibition. Replacement of P
i
by arsenate led to
complete arsenolysis of Xao. Kinetic parameters are
reported for the phosphorolytic and reverse synthetic path-
ways at several selected pH values. Phosphorolysis of
200 l
M
Xao by the human enzyme at pH 5.7 is inhibited by
Guo (IC
50
¼ 10 ± 2 l
M
), Hx (IC
50
¼ 7±1l
M
)andGua
(IC
50
¼ 4.0 ± 0.2 l
M
). With Gua, inhibition was shown to
be competitive, with K
i
¼ 2.0 ± 0.3 l
M
.Bycontrast,Xao
and its products of phosphorolysis (Xan and R1P), were
poor inhibitors of phosphorolysis of Guo, and Xan did not
inhibit the reverse reaction with Gua. Possible modes of
binding of the neutral and anionic forms of Xan and Xao by
mammalian PNPs are proposed. Attention is directed to the
fact that the structural properties of the neutral and ionic
forms of XMP, Xao and Xan are also of key importance in
many otherenzyme systems, such as IMP dehydrogenase,
some nucleic acid polymerases, biosynthesis of caffeine and
phosphoribosyltransferases.
Keywords: Purinenucleoside phosphorylases; xanthine/
xanthosine; enzyme kinetics; enzyme–ligand interactions;
pH-dependence.
The ubiquitous purinenucleoside phosphorylases (PNP,
purine nucleoside phosphorylase ribosyl transferases), cat-
alyse the cleavage (phosphorolysis) of the glycosidic bond
of ribo- and 2¢-deoxyribo- nucleosides in the presence of
inorganic phosphate (P
i
), a reaction reversible with natural
substrates, as follows:
b-nucleoside+P
i
ÀÀ*
)ÀÀ
PNP
purine base
þ a-D-ribose-1-phosphate
In mammalian cells, phosphorolysis is the predominant
reaction, due to coupling with guanase and xanthine
oxidase, leading to stepwise formation of xanthine (Xan)
and, finally, urate. PNP functions in the so-called purine
salvage pathway, wherein the purines liberated by phos-
phorolysis are converted by hypoxanthine-guanine phos-
phoribosyltransferase (HGPRTase) to the monophosphates
of inosine (Ino) and guanosine (Guo).
The natural substrates of the mammalian enzymes are the
6-oxopurine nucleosides, Ino and Guo, and their 2¢-deoxy
counterparts, but not the 6-aminopurine nucleosides, ade-
nosine (Ado) and dAdo. By contrast, all the aforementioned
are substrates for the enzyme from E. coli, a product of
the deoD gene [1], as well as for the enzyme from many
prokaryotes, e.g. S. typhimurium. It was first shown by
Hammer-Jespersen et al. [2] that cultivation of E. coli K-12
cells in the presence of xanthosine (Xao), but no other
nucleoside or base, led to the expression of a second
Correspondence to B. Kierdaszuk, Department of Biophysics,
Institute of Experimental Physics, University of Warsaw,
93
_
ZZwirki i Wigury Street, 02-089 Warsaw, Poland.
Fax: + 48 22 554 0001, Tel.: + 48 22 554 0715,
E-mail: borys@biogeo.uw.edu.pl
Abbreviations: PNP, purinenucleoside phosphorylase; HGPRTase,
hypoxanthine-guanine phosphoribosyltransferase; Xao, xanthosine;
dXao, 2¢-deoxyxanthosine; Xan, xanthine; 6-thio-Xan, 6-thioxan-
thine; 2-thio-Xan, 2-thioxanthine; m
7
Guo, N(7)-methylguanosine;
m
7
-6-thioGuo, N(7)-methyl-6-thioguanosine; R1P, a-
D
-ribose-
1-phosphate; dR1P, 2¢-deoxy-a-
D
-ribose-1-phosphate; NR
+
,
nicotinamide 1-b-
D
-ribose.
Enzymes: PNP (EC 2.4.2.1); E. coli PNP-I (EC 2.4.2.1); E. coli PNP-II
(EC 2.4.2.1); HGPRTase (EC 2.4.2.8), guanase (EC 3.5.4.3); xanthine
oxidase (EC 1.1.3.22); IMP dehydrogenase (EC 1.1.1.205);
nucleoside triphosphate pyrophosphatase (EC 3.6.1.19).
(Received 30 April 2002, revised 27 June 2002,
accepted 5 July 2002)
Eur. J. Biochem. 269, 4048–4057 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03097.x
phosphorylase, a product of the xapA gene, which, in
addition to Ino and Guo, accepts Xao as a substrate but
not Ado. This enzyme was initially referred to as Xao
phosphorylase, subsequently as Ino–Guo phosphorylase;
werefertoitasE. coli PNP-II. It was subsequently partially
purified and some of its properties further characterized [3].
This directed our attention to the specificity of the
mammalian enzymes, earlier reviewed [4,5], but with limited
reference to Xan and Xao as substrates. A more recent
review [6] has redirected attention to this and, amongst
others, to a much earlier report by Friedkin [7]. In this
report, phosphorolysis of dGuo by a partially purified PNP
from rat liver, known to contain guanase, led to the
appearance of the then unknown 2¢-deoxyxanthosine
(dXao), which was isolated in crystalline form. This can
be interpreted by the following reaction sequence:
dGuo + P
i
ÀÀ*
)ÀÀ
PNP
Gua+dR1P
# Guanase
Xan+dRIP
ÀÀ*
)ÀÀ dXao+P
i
Phosphorolysis of Guo by the same enzyme preparation led
to the appearance of Xao, also shown more recently by
Giorgelli et al. [8], who do not refer to the paper by Friedkin
[7]. It is assumed that the rat liver preparation was devoid of
xanthine oxidase activity. In the presence of xanthine
oxidase and an excess of P
i
, the isolated dXao was a
substrate for phosphorolysis, at about 2% of the rate for
dGuo at pH 7.4 [7].
It is rather surprising that, in light of this and other
reports on the substrateproperties of Xao and Xan with
mammalian PNPs [9,10], no account has been taken of their
structures and the pH-dependence of these structures. Both
Ino and Guo, with pK
a
values of 8.8 and 9.2, respectively,
due to dissociation of the N(1)-H, exist predominantly as
the neutral 6-oxo forms at physiological pH. In striking
contrast (see Fig. 1), it was shown long ago that Xao
(pK
a
¼ 5.7) is predominantly a monoanion at physiological
pH, at which Xan (pK
a
¼ 7.7) is an equilibrium mixture of
the neutral and monoanionic species. Moreover, unlike Ino
and Guo, and hypoxanthine (Hx) and guanine (Gua),
where monoanion formation at pH > 8 is due to dissoci-
ation of the N(1)-H [11] so that they are no longer 6-oxo
purines, it is the N(3)-H which dissociates in Xao and Xan
[11–13], so that their monoanions, like the neutral forms, are
still 6-oxopurines (see Fig. 1). This is further supported by
the finding that it is the N(3)-H which is dissociated in the
crystal structure of the monoanion of Xan [14], and that the
pK
a
of 1-methyl-Xao, where only the N(3)-H can dissociate,
is 5.85 [12], close to 5.7 for Xao.
Bearing in mind the physiological significance of Xan and
Xao in the purine salvage pathway and the differences in
structure between the neutral and monoanionic forms of
these relative to those of Hx and Ino, and Gua and Guo, it is
clearly desirable to determine the substrateproperties of the
neutral and monoanionic forms of Xan and Xao. This is also
relevant to the properties of the E. coli PNP-II, referred to
above, which exhibits a marked preference for Xao, as well as
to a number of otherenzyme systems, discussed below.
MATERIALS AND METHODS
Materials
Purine nucleoside phosphorylase from human erythrocytes
and calf spleen (Sigma, St Louis, MO, USA) was further
purified by size-exclusion chromatography, followed by
concentration, as described previously [15]. Specific activi-
ties of the enzymes are given in the footnote to Table 2.
Guo, Ino, formycin B, disodium arsenate, mono- and
disodium phosphate and a-
D
-ribose-1-phosphate (R1P)
were also obtained from Sigma, and Xao and Xan from
Fig. 1. Structures of the neutral and monoanionic forms of hypoxanthine (Hx) and inosine (Ino), guanine (Gua) and guanosine (Guo), and xanthine
(Xan) andxanthosine (Xao). Note that the monoanions of the latter are still 6-oxopurines, like the neutral forms of Gua and Guo, and Hx and Ino.
Ó FEBS 2002 Xanth(os)ine andpurinenucleoside phosphorylases (Eur. J. Biochem. 269) 4049
Serva (Heidelberg, Germany). N-methylated xanthines, and
thioxanthines, were prepared as described previously
[16,17]. The purity of compounds was confirmed by
chromatography and pH-dependent UV absorption spec-
tra. All solutions were prepared with Milli-Q water
(Millipore), using reagents of the highest quality commer-
cially available.
From amongst four commercially available preparations
of Xao, only that from Serva was chromatographically
homogeneous, with pH-dependent UV spectra (Table 1)
consistent with those reported previously [11,12], and the
absence of contaminants further confirmed by
1
Hand
13
C
NMR spectroscopy. This is relevant to earlier reports on the
substrate properties of Xao.
Buffering media, acetate (pH 3.6, 4.5, 5.0 and 5.5), Mes
(pH 6.0 and 6.5), Hepes (pH 7.0, 7.5 and 8.2), Ches (pH 8.5
and 9.0) and Caps (pH 10.0) (Sigma) were selected to avoid
buffer effects on enzyme activity previously noted with Tris
and other buffers [4,15,18,19]. Enzyme activities with these
buffers were unaffected, within experimental error, when
acetate was replaced by Mes at pH 5.0, Mes by Hepes at
pH 6.8, and Hepes by Ches at pH 8.4.
Measurements of pH (± 0.05) were carried out with a
CP315m pH meter (Elmetron, Poland) equipped with a
combination semimicro electrode (Orion, UK) and temper-
ature sensor. UV absorption was monitored with a Kontron
Uvikon 922 recording instrument, fitted with a thermostat-
ically controlled cell compartment, using 1-, 2-, 5- or 10-mm
pathlength cuvettes.
Enzyme kinetics
Phosphorolysis was monitored spectrophotometrically at
25 °Cin50m
M
buffers in the presence of 8 m
M
(substrate
saturation) P
i
, by following the maximal changes in
absorption of the substrate Xao at 242 nm and Guo at
257 nm (Fig. 2) due to formation of Xan and Gua,
respectively. The concentration of P
i
(8 m
M
)ateachpH
was well above its K
m
(< 1 m
M
). The absorption spectra of
each reaction showed isosbestic points, at each pH, e.g. 223,
260 and 279 nm (with Xao), and 240 and 287.5 nm (with
Guo) at pH 5.7, which permitted the monitoring of product
formation in the reaction mixture. The reverse synthetic
reaction was monitored in the presence of 1 m
M
(substrate
saturation) R1P and no P
i
. For pH effects on enzyme
activity, enzyme samples were preincubated at each pH and
Table 1. Spectral properties of compounds.
Compound pK
a
pH k
max
(nm) e
max
(
M
–1
Æcm
)1
)
Xao 5.7
a
3.6 235, 264 8100, 9200
5.7 253 8700
6.0 251, 271 9100, 8100
6.5 249, 276 9700, 8700
9.0 248, 278 10 100, 9000
Xan 7.7
a
3.6 268 10 400
6.0 267 10 300
7.5 271 9200
10.0 241, 278 9000, 9300
2-thio-Xan 5.9
b
8.0 278 15 000
6-thio-Xan 6.5
b
7.0 342 24 600
Guo 9.2 7.0 253 13 700
Gua 9.3 7.0 246 10 700
a
pK
a
values for Xao and Xan were taken from references
[11,12,60], independently confirmed in this study by spectropho-
tometric titration at 25 °C.
b
Determined by spectrophotometric
titration.
Fig. 2. pH-Dependence of relative activities, expressed as initial rates, of PNP from (A, B) human erythrocytes and (C, D) calf spleen, for phos-
phorolysis of 1.2 m
M
Xao (d), and 200 l
M
(pH < 8), 500 l
M
(pH = 8) and 780 l
M
(pH = 8.5) Guo (m), and for the reverse synthetic reaction with
1.2 m
M
(pH < 7) and 1.8 m
M
(pH ‡ 7) Xan (O) and 100 l
M
Gua (m). Activities of both enzymes vs. 200 l
M
Guo and 100 l
M
Gua at pH 7 were
taken as 100%, for the phosphorolytic and synthetic reactions, respectively. Measurements were in 50 m
M
buffers containing 8 m
M
P
i
for
phosphorolysis, and 1 m
M
R1P for the reverse reaction at 25 °C. Reactions for Xao and Xan were monitored spectrophotometrically at 242 nm,
for which values of De were: 4030 (pH 3.6), 4040 (pH 4.5), 4320 (pH 5.0), 4640 (pH 5.7), 4740 (pH 6.0), 4820 (pH 6.5), 4130 (pH 7.0), 3240
(pH 7.6), 1720 (pH 8.1) and 440 (pH 8.5); and for Guo and Gua at 257 nm, with De of 4600 in the pH range 3.6–8.5.
4050 G. Stoychev et al.(Eur. J. Biochem. 269) Ó FEBS 2002
their activities were measured, at concentrations as close
to saturation as possible, with Xao (% 1.5 m
M
), Guo
(% 0.5 m
M
)andP
i
(8 m
M
) for phosphorolysis, and Xan
(% 1.2 m
M
), Gua (% 100 l
M
)andR1P(1m
M
)forthe
reverse reaction. Concentrations of substrates may be
considered as saturated only in the pH range of 5–6 (for
phosphorolysis of Xao) and 5.0–7.5 (for the reverse reaction
with Xan), where they are at least threefold higher than the
appropriate K
m
values (Table 2). Due to low solubility of
Gua and Xan, they were initially dissolved in slightly
alkaline medium and then diluted with buffer to the
appropriate pH. Concentrations of nucleosides and bases
were determined from absorbance measurements, using
molar extinction coefficients (Table 1).
Kinetic constants were determined using the initial rate
method. Initial rates (v) were determined from linear
regression fitting to at least 10 experimental points for the
linear course of the reaction (1–2 min), with an accuracy
of £ 5%. The values of the Michaelis constant (K
m
)and
maximal velocity (V
max
) were determined from nonlinear
regression fitting of the Michaelis–Menten eqn (1) to initial
rates (v) measured for the whole concentration range of
substrate ([S]):
v ¼ V
max
=ð1 þ K
m
=½SÞ ð1Þ
Inhibition constants (K
i
) were calculated using the Dixon
equation for competitive inhibition:
1=v ¼½ðK
m
=[S])ð1 þ [I]=K
i
Þþ1=V
max
ð2Þ
Equation (2) was fitted to initial rates measured at four
concentrations of inhibitor [I] for each substrate concentra-
tion (Fig. 3), and apparent values of K
i
calculated.
RESULTS
Reaction equilibrium for phosphorolysis of Xao
Substrate properties of Xao and Xan for PNPs from
mammalian sources have been reported previously by several
groups [7–10,20], but in each case only at a single arbitrarily
selected pH. These experiments did not take into account the
existence of a mixture of neutral and monoanionic forms,
and with no measurements of the kinetic constants.
The phosphorolytic conversion of 100 l
M
Xao to Xan by
calf spleen PNP was followed in the presence of 8 m
M
P
i
at
pH 5.7, where the population of the neutral form of Xao is
% 50%, and that of the neutral form of Xan is % 100%. The
reaction levels off at about 83% conversion, corresponding
to an equilibrium constant of 0.05. This is not due to enzyme
inactivation, as addition of fresh enzyme at this point was
without effect. Nor is it due to product inhibition, because
the initial rate of the reaction was unaffected in the presence
of 1 m
M
Xan, and the IC
50
of R1P was 1 m
M
. The levelling
off of the reaction must therefore be due to establishment of
equilibrium with the reverse synthetic reaction, confirmed
by addition of 0.25 m
M
R1P, which led to reduction of the
Fig. 3. Dixon plot for the inhibition of phosphorolysis of Xao by Gua
with human PNP at pH 5.7 and 25 °C: (j)290l
M
Xao (d)580l
M
Xao (m) 1160 l
M
Xao. The solid lines represent linear equations fitted
independently using the linear regression method.
Table 2. Kinetic parameters for phosphorolysis of Xao and Guo (in presence of 8 m
M
P
i
), and for the reverse synthetic reaction with Xan and Gua (in
presence of 1 m
M
R1P), for human and calf PNP at various pH values.
human PNP calf PNP
Compound pH
K
m
l
M
V
max
a
%
V
max
/K
m
a
%
K
m
l
M
V
max
a
%
V
max
/K
m
a
%
Xao 5.7 580 ± 150 42 ± 10 0.9 ± 0.2 400 ± 100 126 ± 23 3.5 ± 0.8
6.5 1600 ± 300 18 ± 4 0.14 ± 0.07 1100 ± 100 54 ± 12 0.5 ± 0.3
Guo
b
5.7 91 ± 13 55 ± 5 7 ± 5 46 ± 3 50 ± 5 12 ± 5
6.5 14 ± 2 79 ± 5 68 ± 5 15.9 ± 1.6 79 ± 5 55 ± 5
7.0 12 ± 2
c
100
d
100
d
11 ± 2
e
100
f
100
f
Xan 6.0 380 ± 15 28 ± 5 1.0 ± 0.2 280 ± 20 95 ± 15 2.0 ± 0.5
7.5 425 ± 85 26 ± 5 0.8 ± 0.2 306 ± 40 13 ± 4 0.3 ± 0.1
Gua
g
6.0 29 ± 2 81 ± 6 39 ± 5 11 ± 1 60 ± 6 33 ± 4
7.0 13.8 ± 0.6 100
d
100
d
6.0 ± 1.2
h
100
f
100
f
7.5 22 ± 1 99 ± 2 62 ± 10 9 ± 1 89 ± 5 59 ± 8
a
Values for Xao and Xan are relative to Guo and Gua, respectively, at pH 7.
b
Values of V
max
and V
max
/K
m
at pH 5.7 and pH 6.5 are
relative to those at pH ¼ 7.
c
12 ± 1 l
M
in [31].
d
Values of k
cat
and k
cat
/K
m
are 33 ± 4 s
)1
and 2.8 ± 0.5 s
)1
Æl
M
)1
, and 43 ± 5 s
)1
and
3.1 ± 0.6 s
)1
l
M
)1
for Guo and Gua, respectively (cf. Stoeckler et al. [31]).
e
11 l
M
in [61].
f
Values of k
cat
and k
cat
/K
m
are 31 ± 4 s
)1
and
2.8 ± 0.5 s
)1
l
M
)1
, and 23 ± 3 s
)1
and 3.8 ± 0.7 s
)1
l
M
)1
for Guo and Gua, respectively (cf. Porter [62]).
g
Values of V
max
and V
max
/K
m
at
pH 6.0 and 7.5 are relative to those at pH 7.
h
6±1l
M
in [63].
Ó FEBS 2002 Xanth(os)ine andpurinenucleoside phosphorylases (Eur. J. Biochem. 269) 4051
plateau level to % 53%, and lack of an effect of addition of
30 l
M
Xan. When P
i
was replaced by arsenate, arsenolysis
proceeded at a slower rate, but on prolonged incubation
went virtually to completion. This is because arsenolysis is
not reversible, due to very rapid hydrolysis of a-
D
-ribose-1-
arsenate [21].
pH-dependence of substrate properties
Figure 2 exhibits the substrateproperties of Xao and Xan
with the human and calf enzymes over the pH range 3.6–8.7,
and, for comparison, those of Guo and Gua. Note that,
because of their poorer substrateproperties relative to Guo
and Gua, the concentrations of Xao and Xan employed
were necessarily several-fold higher than those of Guo and
Gua (Fig. 2). Despite this, the concentrations of Xao and
Xan may be considered as saturating at pH values where
these concentrations are several-fold higher than the
appropriate K
m
values (Table 2), i.e. for Xao at pH 5–6,
and for Xan at pH 5–7.5. The use of higher concentrations
of Xan was limited by the low solubility, and by the decrease
in accuracy of reaction rates monitored by small changes
of very high absorbancy of substrates, even with a 1-mm
optical path length.
It is clear from Fig. 2 that, whereas the optimum pH for
both Guo and Gua is in the range 7–8, that for Xao and
Xan is in the range pH 5–6, particularly pronounced for the
calf spleen enzyme. It is of interest, in this context, that with
E. coli PNP-II, the pH profile for Xao (optimum 6.7) has
been shown to overlap those for dGuo (optimum 6.7) and
Guo (optimum 6.9) [22]. With both calf and human
enzymes, phosphorolysis of Xao is optimal at about pH 5
(where the population of the neutral species is % 70%), and
decreases with increase in pH, as compared to an increase
for Guo. This points to the neutral form of Xao as the
preferred substrate, further supported by its high activity
with both enzymes at pH 3.5 (Fig. 2B,D), where it exists
exclusively as the neutral form. The marked decrease in the
rate of phosphorolysis above pH 6, where phosphorolysis
of Guo increases, further suggests that the neutral form of
Xao may be the exclusive substrate.
The same applies to Xan in the reverse synthetic reaction
with both enzymes, the rate of which decreases sharply
above pH 6, at which the monoanionic form appears
(pK
a
¼ 7.7). The pH profiles suggest that the monoanionic
form of Xan is two orders of magnitude weaker as a
substrate than the neutral form.
We then compared the pH-dependence of enzyme activity
for Xao and Xan with a substrate which does not undergo
ionization in the pH range 6–9. Such a substrate is the
cationic nicotinamide riboside (NR
+
), which, like the cation
of m
7
Guo [23], undergoes nonreversible phosphorolysis by
the enzymes from mammalian sources and E. coli [24]. V
max
and V
max
/K
m
for NR
+
, which is exclusively in the cationic
form (pK
a
% 11.9), is unchanged over the pH range 7–10
[24]. It follows that the pH-dependence of reaction rates for
Xao and Xan in the pH range 6–9 should reflect changes in
substrate properties due to ionization of the base moiety.
Substrate properties of thioxanthines
The apparent substrateproperties of the monoanion of
xanthine directed our attention to 2-thio-Xan and 6-thio-
Xan, both of which would be expected to be more acidic
than the parent Xan, and hence with higher populations of
the monoanions at physiological pH. We have confirmed
this by spectrophotometric titration, which gave pK
a
values
of 5.9 ± 0.1 for 2-thio-Xan and 6.5 ± 0.1 for 6-thio-Xan.
For 6-thio-Xan the predominant tautomer of the neutral
form was identified by means of UV spectroscopy in
aqueous medium and by NMR spectroscopy in dimethyl-
sulfoxide-water [16,17] as the 6-thione-2-oxo-N(7)-H. To
our knowledge there are no experimental data on the
structure of the anionic species, but a recent theoretical
study [25] suggests that the neutral form of 6-thio-Xan is
6-thione-2-oxo-N(7)-H, and that monoanion formation
involves dissociation of the N(3)-H, as for the parent Xan
(Fig. 1). Furthermore, it points to 6-oxo-2-thione-N(9)-H as
being more stable than 6-oxo-2-thione-N(7)-H in aqueous
medium [26].
Hitherto, thioxanthines and their nucleosides have not
been examined as potential substrates of PNP, and studies
of their substrates’ propertieswithother enzymes have not
considered their physico-chemical properties, notwithstand-
ing that 6-thio-Xan is an intermediate in the metabolism of
thiopurines [27]. These compounds are considered as
potential antitumour agents [28], and 6-thio-Xan is a
prodrug in gene therapy [29].
To compare the substrateproperties of 6-thio-Xan and
Xanwithbothenzymes,weestimatedDe for the following
conversion
6-thio-Xan
ÀÀ*
)ÀÀ 6-thio-Xao
from the differences between the absorption spectra of
6-thio-Xan, and those reported for 6-thio-Xao [30], at pH 2,
7 and 11. At pH 5, where the population of the neutral form
is > 90%, conversion of 6-thio-Xan (400 l
M
) to 6-thio-Xao
(De % 4000 at k
obs
¼ 355 nm) in the presence of 1 m
M
R1P
was 10-fold slower than for the parent Xan. Raising the pH
to 8.2, where the population of the monoanion of 6-thio-
Xan is % 98% (as compared to % 75% for Xan) reduced its
reaction rate, which at this pH was similar to that for Xan,
further pointing tosubstrate activity of the monoanion.
This is consistent with the proposed existence of the neutral
form as 6-thio-2-oxo, and dissociation of the N(3)-H to
form the monoanion [25], as for the parent Xan monoanion.
With 2-thio-Xan, quantitative measurements of enzyme
activity were not possible, because the UV absorption
spectra of its nucleoside are unknown. However, spectral
changes at pH ¼ 5(% 90% neutral form), and at pH 8
(% 100% anion), in the presence of the enzymeand R1P,
were barely detectable, pointing to its being a very feeble
substrate, if at all. The poor, if any, substrateproperties of
2-thio-Xan clearly call for an investigation of the structures
of its neutral and monoanionic species.
Kinetic constants
Table 2 presents several kinetic constants for Xao and Xan
at selected pH values, and the corresponding constants for
Guo and Gua, with the human and calf spleen enzymes.
Surprisingly, the V
max
for Xao at pH 5.7 with the calf, but
not human, enzyme is 2.5-fold higher than for Guo.
However, it should be noted that the K
m
values for both
Xao and Xan are very high relative to those for Guo and
4052 G. Stoychev et al.(Eur. J. Biochem. 269) Ó FEBS 2002
Gua, accounting in large part for the lower rate constants,
V
max
/K
m
, of the former in both the phosphorolytic and
reverse reaction pathways.
The V
max
/K
m
for phosphorolysis of Xao at pH 5.7 is,
with the human enzyme, 13% of that for Guo, andwith the
calf enzyme, 29% that for Guo. These values decrease
dramatically in going from pH 5.7 to 6.5, i.e. with a large
increase in population of the monoanionic species of Xao,
indicating that the enzymes highly prefer the neutral form.
Similarly, the V
max
for Xan in the reverse synthetic reaction
with the calf enzyme decreases sevenfold in going from
pH 6 to 7.5, i.e. with an increase in population of the
monoanionic form, accounting for the sevenfold lower rate
constant at pH 7.5. By contrast, for Xan in the reverse
synthetic reaction with the human enzyme, both V
max
and
V
max
/K
m
are barely affected by an increase of pH from 6 to
7.5, in line with the smaller effect of pH, in this pH range, on
the V
max
and V
max
/K
m
for Gua in the reverse reaction with
the human, than with the calf, enzyme (Table 2).
Competition and product inhibition
Possible competition between Xao and Guo was investi-
gated by monitoring phosphorolysis of each by human PNP
in a medium containing 200 l
M
Xao and 10 l
M
Guo, in the
presence of 8 m
M
P
i
at pH 5.7. Phosphorolysis of Guo was
followed at 260 nm, where there is an isosbestic point for the
interconversion
Xao
ÀÀ*
)ÀÀ Xan ðDe ¼ 0Þ
Guo
ÀÀ*
)ÀÀ Gua ðDe ¼ 4600Þ
Phosphorolysis of Xao was monitored at 287.5 nm, the
isosbestic point for
Guo
ÀÀ*
)ÀÀ Gua ðDe ¼ 0Þ
Xao
ÀÀ*
)ÀÀ Xan ðDe ¼ 2200Þ
The rate of phosphorolysis of 10 l
M
Guo at pH 5.7 was
only minimally affected in the presence of 200 l
M
Xao. As
the latter is a 1 : 1 mixture of the neutral and monoanionic
forms at this pH, it follows that both are poor inhibitors. By
contrast, the initial rate of phosphorolysis of 200 l
M
Xao at
this pH was inhibited by about 50% in the presence of
10 l
M
Guo, and this inhibition was markedly accentuated
as the reaction proceeded, pointing to the involvement of
some product of phosphorolysis. Both Xan and R1P were
very poor inhibitors, with IC
50
>1m
M
. However, Gua
proved to be a good inhibitor of phosphorolysis of Xao by
human PNP (IC
50
% 4 l
M
), as was Hx (IC
50
% 7 l
M
). In
the case of Gua, inhibition was shown to be competitive
(Fig. 3), with K
i
¼ 2.0 ± 0.3 l
M
.Itistobeexpectedthat,
at pH > 6.5, inhibition will be more pronounced because
of the threefold higher K
m
for Xao, whereas K
m
values of
Guo and Gua are unchanged (Table 2). However, the high
K
m
at pH ¼ 6.5 proved to be an obstacle to measurement of
K
i
for Gua at this pH.
The reverse reaction for human PNP with 10 l
M
Gua
and 1 m
M
(saturated) R1P was not affected in the presence
of 100 l
M
Xan at pH 7, where the latter is a 6 : 1 mixture of
the neutral and monoanionic forms. By contrast, Xan was a
good inhibitor (IC
50
% 20 l
M
) of the reverse reaction, with
human PNP and 10 l
M
Hx (i.e. at its K
m
value [31]) and
1m
M
R1P. This is consistent with the finding of Krenitsky
et al. [32] that the reverse reaction for Hx with human PNP
is inhibited by Xan with K
i
¼ 40 l
M
.
Formycin B, a structural analogue of Ino, is a weak
inhibitor of phosphorolysis of Ino by the human and calf
enzymes at pH 7, with K
i
% 100 l
M
, and an even weaker
inhibitor of both Ino and Xao phosphorolysis by E. coli
PNP-II [3], with K
i
% 300 l
M
. We have examined the effect
of formycin B on phosphorolysis of Xao and Guo by the
human enzyme at pH 5.7, at substrate concentrations
comparable to their K
m
values, 500 l
M
and 90 l
M
, respec-
tively. This led to IC
50
values of 160 ± 30 l
M
versus Xao
and 500 ± 100 l
M
versus Guo, and shows that more
effective inhibition of Xao correlates with its lower activity
as substrate.
All four N-monomethyl xanthines were found to be very
poor, or barely detectable, inhibitors of phosphorolysis by
both the calf and human enzymes at pH 5.7, where the
1-methyl-, 3-methyl- and 7-methyl- xanthines are predomi-
nantly in the neutral forms, and 9-methylxanthine
(pK
a
% 6.3) is a mixture of neutral and monoanionic
species [11]. Krenitsky et al. [32] had previously reported
that all of these were very poor inhibitors of the reverse
synthetic pathway by the human enzyme at pH 7.2, where
they are all mixtures of neutral and monoanionic forms. It
follows that both the neutral and monoanionic species of all
four monomethyl xanthines are very poor inhibitors of both
the phosphorolytic and synthetic pathways, and that
dimethyl xanthines should also be poor inhibitors, as found.
DISCUSSION
Comparison with earlier data
Bearing in mind differences between enzymes from different
sources, as shown here between the human and calf
enzymes, it is instructive to note that our results are in
general accord with data reported earlier, but only at single
pH values, e.g. for (a) phosphorolysis of Xao at pH ¼ 6by
human erythrocytic PNP [10], (b) synthesis of Xao from
Xan by bovine liver PNP at pH ¼ 8 [9], (c) synthesis of Xao
from Xan by the calf spleen enzyme at pH ¼ 7[20],and(d)
phosphorolysis of dXao and Xao by rat liver PNP at
pH ¼ 7.4 [7].
Possible modes of binding of Xao and Xan by PNP
Information now available, largely from crystallographic
studies, of the modes of binding of Hx and Gua, and their
nucleosides, as well as nucleoside analogue inhibitors, by the
PNPs from various sources [33–37], permits inferences of
modes of binding of Xao and Xan, for which no experi-
mental data are available.
TheactivecenterofE. coli PNP-I [37], which does not
accept Xao and Xan, differs from those of the mammalian
enzymes in that it does not contain the Glu201 of the latter.
This residue is proposed to play a key role in the catalytic
process via Ôtwo-hydrogen bond bindingÕ of the Glu201O
e1
and Glu201O
e2
to the C(2)-NH
2
and the N(1)-H of Gua [or
the N(1)-H of Hx] thus stabilizing the intermediate state of
the base [33–36]. This is in line with the preference of the
mammalian enzymes for the neutral 6-oxo forms of Gua
and Guo [38–40], Hx and Ino, and the cationic 6-oxo forms
Ó FEBS 2002 Xanth(os)ine andpurinenucleoside phosphorylases (Eur. J. Biochem. 269) 4053
of m
7
Guo [23] and m
7
-6-thioGuo [41]. With the mono-
anions of Gua and Guo, and the zwitterions of m
7
Guo and
its m
7
-6-thioGuo, there will be electrostatic repulsion
between the negative charge on N(1) and the anionic form
of the Glu201 carboxyl in the active site of the mammalian
enzymes (and E. coli PNP-II), which is absent in the active
site of PNP-I.
The above suggests that, for binding of Xan and Xao in
the active sites of the mammalian enzymes (and E. coli
PNP-II), the absence of dissociation of the N(1)-H is of key
importance for the substrateproperties of their mono-
anions, inasmuch as dissociation of the N(3)-H still permits
interaction of the N(1)-H with either the neutral or anionic
forms of Glu201 carboxyl (Fig. 4), and hence their substrate
properties, as observed. Dissociation of these protons
reduced substrate activity at slightly alkaline pH, similarly
to that observed at pH < 5, where protonation of His64
also led to a significantly reduced enzyme efficiency (Fig. 2).
The proposed modes of binding of Xan by calf and
human enzymes should also incorporate data showing that,
in aqueous medium, Xan exists as a mixture of the N(7)-H
and N(9)-H tautomeric forms [13]. This is often overlooked
in analysis of binding and reverse reactions with Gua and
Hx, with only the N(9)-H tautomer taken into account,
because of its structural similarity to natural purine
nucleosides. One possible mode of binding of the N(9)-H
form of Xan is similar to that shown for binding of Xao
(Fig. 4), originally proposed by Mao et al.[33]forInoand
sulfate in the active site of bovine spleen PNP, although in
their PDB entry (1A9S), Asn243 is rotated in such a way
that Asn243N
d
donates a hydrogen to O
6
of the base.
Involvement of O
6
in binding to the enzyme from Cellulo-
monas, the properties of which are similar to those of the
mammalian enzymes, was also proposed by Tebbe et al.
[42], based on the structure of its complex with 8-iodogua-
nine and sulfate (or phosphate). This is more feasible with
the N(7)-H tautomer, further supported by data on the
ternary complex bovine PNP/9-deazaIno/P
i
[35], and some
9-deazaGuo inhibitors, where N(7) is protonated, complexed
with human erythrocyte PNP [43]. A similar pattern was
observed for the ternary complex of human PNP with the
transition-state analogue inhibitor immucillin-H and P
i
[34],
again pointing to possible involvement of the N(7)-H form
of purine bases in the reverse reaction. We propose that the
N(7)-H tautomers of the neutral and ionic forms of Xan are
preferentially bound by the active sites of the human and
calf enzymes, and the N(7)-H donates a hydrogen to
Asn243O
d
, while the Asn243N
d1
donates a hydrogen to the
exocyclic O
6
of the neutral and ionic forms of Xan (Fig. 4).
Additionally, a bridging water molecule between O
6
and
Glu201O
e2
(not shown) could also be present here, as
observed for purines andpurine nucleosides in the active site
of human erythrocyte PNP [40], for Hx in the binary
complex with the calf spleen enzyme [36], and for the ternary
complex bovine-PNP/immucillin-H/P
i
[34]. By contrast, in
E. coli PNP-I, Asn is replaced by Asp204, so that binding of
the ligand is additionally dependent on ionization of the Asp
side chain, which would then electrostatically repel the
anions of Xan and Gua, irrespective of the site of
dissociation in these purines. Furthermore a bridging water
molecule is not observed, e.g. in the formycin B complex
with E. coli PNP-I [37], which in the case of mammalian
enzymes may be involved in enzyme-ligand binding and/or
the enzymatic reaction.
Fig. 4. Proposed models of binding by mam-
malian PNPs of the neutral (A, B, D, E) and
anionic (C, F) forms of xanthine (A–C) and
xanthosine (D–F), based on the enzyme–ligand
interactions observed in the crystal structures of
immucillin-H [34] and hypoxanthine [33,36]
with calf spleen PNP, and 5¢-iodo-9-deazaino-
sine with human PNP [35]. Note proposed
binding with the neutral (A, D) and anionic
(B, C, E, F) forms of the Glu201 carboxylate.
See text for further details.
4054 G. Stoychev et al.(Eur. J. Biochem. 269) Ó FEBS 2002
In line with the above, we suggest that both the neutral
and anionic forms of the Glu201 carboxyl hydrogen bonds
the N(1)-H of Xan and Xao, irrespective of the ionization of
N(3)-H (Fig. 4), and together with the interactions main-
tained by Asn243, play a key role in transition state
formation, as well as in phosphorolysis of Xao and the
reverse reaction with Xan.
Relevance tootherenzyme systems
The pK
a
values, and unique structures of the monoanions
of Xao and Xan, as well as of XMP [12], are of equal
relevance in otherenzyme systems, for which they are
substrates or intermediates. One case in point is IMP
dehydrogenase, the rate-limiting enzyme in the de novo
synthesis of guanine nucleotides, which catalyses the NAD-
dependent oxidation of IMP to XMP, which is then
converted to GMP [44]. A novel nucleoside triphosphate
pyrophosphatase from the thermophilic Methanococcus
jannaschii has been reported as highly specific for the
noncanonical nucleotides ITP and XTP, even at high
alkaline pH [45], where both exist exclusively as mono-
anions, albeit with different structures.
The biosynthesis of caffeine, recently extensively reviewed
by Ashihara & Crozier [46], proceeds through a number of
enzymatic steps involving as intermediates Xan and Xao,
XMP and m
7
XMP, followed by cleavage of the latter and
stepwise methylation of the liberated N(7)-methylxanthine
to give N(1),N(3),N(7)-trimethylxanthine (caffeine).
Particularly relevant are the purine phospho-
ribosyltransferases, which function in the salvage pathway
by addition of a preformed purine base to the a–carbon of
a–
D
-phosphoribosylpyrophosphate to generate purine
nucleotides. These include a family of enzymes specific for
6-oxopurines. The human enzyme accepts Hx and Gua, but
only very minimally Xan [47,48]. E. coli contains two such
enzymes, one with a preference for Hx, the other for Gua
and Xan [49]. Parasitic protozoa, which are incapable of
de novo synthesis of purine nucleotides, express a unique
complement of purine salvage enzymes; for example
Leishmania donovani possesses one such enzymewith a
marked preference for Xan [50,51].
The X-ray structures of many of these enzymes in
complexes with 6-oxopurines or their nucleotides, including
Xan and XMP, have been reported, in all instances with the
explicit assumption that the xanthine moiety is uniquely 2,6-
dioxo. However, whereas the modes of binding of Hx and
Guaintheforwardreaction,andIMPandGMPinthe
reverse reaction, have been reasonably well assigned, the
situation is less clear for the modes of binding of Xan and
XMP [49]. It is conceivable that, in the crystal structures,
dissociation of the xanthine N(3)-H is blocked. However,
the resolution of the crystal structures is insufficient to
distinguish between a C¼OandC-O
–
.
We are aware of only one enzyme system, xanthine
oxidase, where attention was directed to possible substrate
properties of the monoanion. On the basis of kinetic and
pH-titration studies, it was proposed that the neutral forms
of Xan [52] and 1-Me-Xan [53] are the preferred substrates,
but with the erroneous assumption that monoanion
formation involves dissociation of an imidazole proton.
These findings do not unequivocally exclude weak substrate
properties of the monoanions.
The foregoing would be incomplete without at least
passing reference to current efforts to develop noncanon-
ical base pairs for replication [54] and transcription
[55,56]. The 5¢-triphosphates of Xao (XTP and dXTP)
have been widely employed for this purpose, and are
complementarily incorporated into RNA and DNA by
some polymerases with moderate selectivity. However, the
Xan moiety has been assumed to be in the neutral 2,6-
diketo form, notwithstanding that it was shown long ago
that poly(xanthylate) forms multistranded helices with
different structures in acid and alkaline media, related to
dissociation of the N(3)-H of Xao [57,58]. This is further
confirmed by X-ray diffraction of fibers, which form one
helix at acid pH with the Xan residues in the neutral
form, and another at pH 8 with dissociation of the N(3)-
H of the Xan residues [59].
ACKNOWLEDGEMENTS
We are indebted to Prof Wolfgang Pfleiderer (University of Konstanz,
Germany) for several authentic samples of N-methyl xanthines. This
investigation was supported by the State Committee for Scientific
Research (KBN, Grant no. 6P04A03812, and partially BW 1482/BF
and BST 661/BF); and by an International Research Scholar’s award
of the Howard Hughes Medical Institute (Grant No. HHMI 75195–
543401). G. S. is also indebted for support to KBN (Grant no.
6PO4A03813), andto the Fellowship Program of the Institute of
Biochemistry and Biophysics.
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Ó FEBS 2002 Xanth(os)ine andpurinenucleoside phosphorylases (Eur. J. Biochem. 269) 4057
. Xanthosine and xanthine Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems Gerasim Stoychev 1 , Borys Kierdaszuk 1 and David Shugar 1,2 1 Department. aqueous medium [26]. Hitherto, thioxanthines and their nucleosides have not been examined as potential substrates of PNP, and studies of their substrates’ properties with other enzymes have not considered. erythrocytes and calf spleen with both Xao and Xan is in the range 5–6, whereas those with Guo and Gua, and Ino and Hx, are in the range 7–8. The pH-dependence of substrate properties of Xao and Xan