Báo cáo khoa học: Xanthosine and xanthine Substrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems docx

Xanthosine and xanthineSubstrate properties with purine nucleoside phosphorylases, and relevance to other enzyme systems Gerasim Stoychev1, Borys Kierdaszuk1and David Shugar1,2 1 Departm

Xanthosine and xanthine

Substrate properties with purine nucleoside phosphorylases, and relevance

to other enzyme systems

Gerasim Stoychev1, Borys Kierdaszuk1and David Shugar1,2

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 purine nucleoside phosphorylases (PNP) of

mammalian origin have been reported previously, but only

at a single arbitrarily selected pH and with no kinetic

con-stants Additionally, studies have not taken into account the

fact that, at physiological pH, Xao (pKa¼ 5.7) is a

mono-anion, while Xan (pKa¼ 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 (pKa¼ 6.5 ± 0.1), but not of

2-thioxan-thine (pKa¼ 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 Pi 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 lMXao by the human enzyme at pH 5.7 is inhibited by Guo (IC50¼ 10 ± 2 lM), Hx (IC50¼ 7 ± 1 lM) and Gua (IC50¼ 4.0 ± 0.2 lM) With Gua, inhibition was shown to

be competitive, with Ki¼ 2.0 ± 0.3 lM By contrast, 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 other enzyme systems, such as IMP dehydrogenase, some nucleic acid polymerases, biosynthesis of caffeine and phosphoribosyltransferases

Keywords: Purine nucleoside phosphorylases; xanthine/ xanthosine; enzyme kinetics; enzyme–ligand interactions; pH-dependence

The ubiquitous purine nucleoside 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 (Pi), a reaction reversible with natural substrates, as follows:

b-nucleoside+Pi *)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 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 _Z Zwirki 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, purine nucleoside phosphorylase; HGPRTase,

hypoxanthine-guanine phosphoribosyltransferase; Xao, xanthosine;

dXao, 2¢-deoxyxanthosine; Xan, xanthine; 6-thio-Xan,

6-thioxan-thine; 2-thio-Xan, 2-thioxan6-thioxan-thine; 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)

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;

w e refer to it as E 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 + Pi *)PNPGua+dR1P

# Guanase Xan+dRIP *) dXao+Pi

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 Pi, 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 substrate properties 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 pKavalues 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 (pKa¼ 5.7) is predominantly a monoanion at physiological

pH, at which Xan (pKa¼ 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

pKaof 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 substrate properties 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 other enzyme systems, discussed below

M A T E R I A L S A N D M E T H O D S

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) and xanthosine (Xao) Note that the monoanions of the latter are still 6-oxopurines, like the neutral forms of Gua and Guo, and Hx and Ino.

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 by1H and13C

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

25C in 50 mMbuffers in the presence of 8 mM(substrate saturation) Pi, 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 Pi (8 mM) at each pH was well above its Km(< 1 mM) 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 mM(substrate saturation) R1P and no Pi 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–1Æcm)1)

6.0 251, 271 9100, 8100 6.5 249, 276 9700, 8700 9.0 248, 278 10 100, 9000

10.0 241, 278 9000, 9300

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.

their activities were measured, at concentrations as close

to saturation as possible, with Xao ( 1.5 mM), Guo

( 0.5 mM) and Pi (8 mM) for phosphorolysis, and Xan

( 1.2 mM), Gua ( 100 lM) and R1P (1 mM) for the

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 Kmvalues (Table 2) Due to lowsolubility 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 (Km) and

maximal velocity (Vmax) 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]):

v¼ Vmax=ð1 þ Km=½SÞ ð1Þ Inhibition constants (Ki) were calculated using the Dixon

equation for competitive inhibition:

1=v¼ ½ðKm=[S])ð1 þ [I]=KiÞ þ 1=Vmax 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 Kicalculated

R E S U L T S

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 lMXao to Xan by calf spleen PNP was followed in the presence of 8 mMPiat

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 mMXan, and the IC50of R1P was 1 mM 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 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) 290 l M Xao (d) 580 l 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.

K m

l M

V maxa

%

V max /K ma

%

K m

l M

V maxa

%

V max /K ma

%

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)1and 2.8 ± 0.5 s)1Æl M )1 , and 43 ± 5 s)1and 3.1 ± 0.6 s)1l M )1 for Guo and Gua, respectively (cf Stoeckler et al [31]).e11 l M in [61].fValues of k cat and k cat /K m are 31 ± 4 s)1and 2.8 ± 0.5 s)1l M )1 , and 23 ± 3 s)1and 3.8 ± 0.7 s)1l 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 ± 1 l M in [63].

plateau level to 53%, and lack of an effect of addition of

30 lMXan When Piwas 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 substrate properties 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 substrate properties 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 Kmvalues (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 coliPNP-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

(pKa¼ 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 m7Guo [23], undergoes nonreversible phosphorolysis by

the enzymes from mammalian sources and E coli [24] Vmax

and Vmax/Kmfor NR+, which is exclusively in the cationic

form (pKa 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 substrate properties 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 pKavalues

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’ properties with other 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 substrate properties of 6-thio-Xan and Xan w ith both enzymes, w e estimated De for the following conversion

6-thio-Xan *) 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 lM) to 6-thio-Xao (De 4000 at kobs¼ 355 nm) in the presence of 1 mMR1P 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 to substrate 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 pH¼ 5 ( 90% neutral form), and at pH 8 ( 100% anion), in the presence of the enzyme and R1P, were barely detectable, pointing to its being a very feeble substrate, if at all The poor, if any, substrate properties 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 Vmaxfor 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 Kmvalues for both Xao and Xan are very high relative to those for Guo and

Gua, accounting in large part for the lower rate constants,

Vmax/Km, of the former in both the phosphorolytic and

reverse reaction pathways

The Vmax/Kmfor phosphorolysis of Xao at pH 5.7 is,

with the human enzyme, 13% of that for Guo, and with 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 Vmaxfor 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 Vmaxand

Vmax/Kmare 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 Vmaxand Vmax/Kmfor 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 lMXao and 10 lMGuo, in the

presence of 8 mMPiat pH 5.7 Phosphorolysis of Guo was

followed at 260 nm, where there is an isosbestic point for the

interconversion

Xao *) Xan ðDe ¼ 0Þ

Guo *) Gua ðDe ¼ 4600Þ

Phosphorolysis of Xao was monitored at 287.5 nm, the

isosbestic point for

Guo *) Gua ðDe ¼ 0Þ

Xao *) Xan ðDe ¼ 2200Þ

The rate of phosphorolysis of 10 lM Guo at pH 5.7 was

only minimally affected in the presence of 200 lMXao 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 lMXao at

this pH was inhibited by about 50% in the presence of

10 lMGuo, 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 IC50> 1 mM However, Gua

proved to be a good inhibitor of phosphorolysis of Xao by

human PNP (IC50 4 lM), as was Hx (IC50 7 lM) In

the case of Gua, inhibition was shown to be competitive

(Fig 3), with Ki¼ 2.0 ± 0.3 lM It is to be expected that,

at pH > 6.5, inhibition will be more pronounced because

of the threefold higher Kmfor Xao, whereas Kmvalues of

Guo and Gua are unchanged (Table 2) However, the high

Kmat pH¼ 6.5 proved to be an obstacle to measurement of

Kifor Gua at this pH

The reverse reaction for human PNP with 10 lMGua

and 1 mM(saturated) R1P was not affected in the presence

of 100 lMXan at pH 7, where the latter is a 6 : 1 mixture of

the neutral and monoanionic forms By contrast, Xan was a

good inhibitor (IC50 20 lM) of the reverse reaction, with

human PNP and 10 lMHx (i.e at its K value [31]) and

1 mMR1P 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 Ki¼ 40 lM 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 Ki 100 lM, and an even weaker inhibitor of both Ino and Xao phosphorolysis by E coli PNP-II [3], with Ki 300 lM 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 Kmvalues, 500 lMand 90 lM, respec-tively This led to IC50values of 160 ± 30 lMversus Xao and 500 ± 100 lM 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 (pKa 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

D I S C U S S I O N

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 pH¼ 6 by human erythrocytic PNP [10], (b) synthesis of Xao from Xan by bovine liver PNP at pH¼ 8 [9], (c) synthesis of Xao from Xan by the calf spleen enzyme at pH¼ 7 [20], and (d) phosphorolysis of dXao and Xao by rat liver PNP at

pH¼ 7.4 [7]

Possible modes of binding of Xao and Xan by PNP Information nowavailable, 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

The active center of E 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 two-hydrogen bond binding of the Glu201Oe1 and Glu201Oe2to the C(2)-NH2and 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

of m7Guo [23] and m7-6-thioGuo [41] With the

mono-anions of Gua and Guo, and the zwitterions of m7Guo and

its m7-6-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 substrate properties 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] for Ino and

sulfate in the active site of bovine spleen PNP, although in

their PDB entry (1A9S), Asn243 is rotated in such a way

that Asn243Nd donates a hydrogen to O6 of the base

Involvement of O6in 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/Pi[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 Pi[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 Asn243Od, while the Asn243Nd1donates a hydrogen to the exocyclic O6of the neutral and ionic forms of Xan (Fig 4) Additionally, a bridging water molecule between O6 and Glu201Oe2 (not shown) could also be present here, as observed for purines and purine 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/Pi[34] By contrast, in

E coliPNP-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.

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 to other enzyme systems

The pKavalues, and unique structures of the monoanions

of Xao and Xan, as well as of XMP [12], are of equal

relevance in other enzyme 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 m7XMP, 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 novosynthesis of purine nucleotides, express a unique

complement of purine salvage enzymes; for example

Leishmania donovani possesses one such enzyme with 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

Gua in the forw ard reaction, and IMP and GMP in the

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 C¼O and C-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]

A C K N O W L E D G E M E N T S

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), and to the Fellowship Program of the Institute of Biochemistry and Biophysics.

R E F E R E N C E S

1 Buxton, R.S., Hammer-Jespersen, K & Valentin-Hansen, P (1980) A second purine nucleoside phosphorylase in Escherichia coli K-12 I Xanthosine phosphorylase regulatory mutants iso-lated as secondary-site revertants of a deoD mutant Mol Gen Genet 179, 331–340.

2 Hammer-Jespersen, K., Buxton, R.S & Hansen, T.D (1980) A second purine nucleoside phosphorylase in Escherichia coli K-12.

II Properties of xanthosine phosphorylase and its induction by xanthosine Mol Gen Genet 179, 341–348.

3 Koszalka, G.W., Vanhooke, J., Short, S.A & Hall, W.W (1988) Purification and properties of inosine-guanosine phosphorylase from Escherichia coli K-12 J Bacteriol 170, 3493–3498.

4 Parks, E Jr & Agrawal, R.B (1972) Purine nucleoside phos-phorylase In The Enzymes, 7 (Boyer, P.D., ed.), pp 483–514 Academic Press, NewYork.

5 Stoeckler, J.D (1984) Purine nucleoside phosphorylase: a target for chemotherapy In Developments in Cancer Chemotherapy (Glazer, R.I., ed.), pp 35–60 CRC Press, Boca Raton.

6 Bzowska, A., Kulikowska, E & Shugar, D (2000) Purine nucleoside phosphorylases: properties, functions, and clinical aspects Pharmacol Ther 88, 349–425.

7 Friedkin, M (1952) Enzymatic synthesis of desoxyxanthosine by the action xanthosine phosphorylase in mammalian tissue J Am Chem Soc 74, 112–115.

8 Giorgelli, F., Bottai, C., Mascia, L., Scolozzi, C., Camici, M & Ipata, P.L (1997) Recycling of alpha- D -ribose 1-phosphate for nucleoside interconversion Biochim Biophys Acta 1335, 6–22.

9 Korn, E.D & Buchanan, J.M (1955) Biosynthesis of the purines

VI J Biol Chem 217, 183–191.

10 Tsuboi, K.K & Hudson, P.B (1957) Enzymes of the human erythrocytes II Purine nucleoside phosphorylase, specific properties J Biol Chem 224, 889–897.

11 Cavalieri, L., Fox, J., Stone, A & Chang, N (1954) On the nature

of xanthine and substituted xanthines in solution J Am Chem Soc 76, 1119–1122.

12 Roy, K.B & Miles, H.T (1983) Tautomerism and ionization of

Xanthosine Nucleosides Nucleotides 2, 231–242.

13 Shugar, D & Psoda, A (1990) Numerical data and functional

relationships in science and technology In Biophysics of Nucleic

Acids, Landoldt-Bo¨rnstein New Series VII/Id (Saenger, W., ed.),

pp 308–348 Springer-Verlag, Berlin.

14 Mizuno, H., Fujiwara, T & Tomita, K (1969) The crystal and

molecular structure of the sodium salt of xanthine Bull Chem.

Soc Japan 42, 3099–3105.

15 Kierdaszuk, B., Modrak-Wo´jcik, A & Shugar, D (1997) Binding

of phosphate and sulfate anions by purine nucleoside

phospho-rylase from E coli: ligand-dependent quenching of enzyme

in-trinsic fluorescence Biophys Chem 63, 107–118.

16 Lichtenberg, D., Bergman, F & Neimann, Z (1971) Tautomeric

forms and ionisation processes in xanthine and its N-methyl

derivatives J Chem Soc C 1971, 1676–1682.

17 Twanmoh, L.M., Wood, H.B Jr & Driscoll, J.S (1973) NMR

spectral characteristics of N-H protons in purine derivatives.

J Heterocycl Chem 10, 187–190.

18 Ropp, P.A & Traut, T.W (1991) Purine nucleoside

phospho-rylase Allosteric regulation of a dissociating enzyme J Biol.

Chem 266, 7682–7687.

19 Kierdaszuk, B., Modrak-Wo´jcik, A., Wierzchowski, J & Shugar,

D (2000) Formycin A and its N-methyl analogues, specific

inhibitors of E coli purine nucleoside phosphorylase (PNP):

induced tautomeric shifts on binding to enzyme, and

enzyme fi ligand fluorescence resonance energy transfer Biochim.

Biophys Acta 1476, 109–128.

20 Krenitsky, T.A (1967) Purine nucleoside phosphorylase: kinetics,

mechanism, and specificity Mol Pharm 3, 526–536.

21 Kline, P.C & Schramm, V.L (1993) Purine nucleoside

phos-phorylase Catalytic mechanism and transition-state analysis of

the arsenolysis reaction Biochemistry 32, 13212–13219.

22 Bezirjian, KhO., Kocharian, Sh.M & Akopyan, Zh.I (1986)

Isolation of a hexameric form of purine nucleoside phosphorylase

II from E coli, Comparative study of trimeric and hexameric

forms of the enzyme Biokhimia 51, 1085–1092.

23 Stoychev, G., Kiedaszuk, B & Shugar, D (2001) Interaction of

Escherichia coli purine nucleoside phosphorylase (PNP) with the

cationic and zwitterionic forms of the fluorescent substrate

N(7)-methylguanosine Biochim Biophys Acta 1544, 74–88.

24 Wielgus-Kutrowska, B., Kulikowska, E., Wierzchowski, J.,

Bzowska, A & Shugar, D (1997) Nicotinamide riboside, an

unusual, non-typical, substrate of purified purine-nucleoside

phosphorylases Eur J Biochem 243, 408–414.

25 Civcir, P.U (2001) Tautomerism of 6-thioxanthine in gas and

aqueous phases using AM1 and PM3 methods J Chem Phys.

114, 1582–1588.

26 Civcir, P.U (2001) Tautomerism of 2-thioxanthine in the gas and

aqueous phases using AM1 and PM3 methods J Mol Struct.

(Teochem.) 546, 163–173.

27 Keuzenkamp-Jansen, C.W., De Abreu, R.A., Bokkerink, J.P.,

Lambooy, M.A & Trijbels, J.M (1996) Metabolism of

intravenously administered high-dose 6-mercaptopurine with and

without allopurinol treatment in patients with non-Hodgkin

lymphoma J Pediatric Hematol./Oncol 18, 145–150.

28 Tamiya, T., Ono, Y., Wei, M.X., Mroz, P.J., Moolten, F.L &

Chiocca, E.A (1996) Escherichia coli gpt gene sensitizes rat glioma

cells to killing by 6-thioxanthine or 6-thioguanine Cancer Gene

Ther 3, 155–162.

29 Ono, Y., Ikeda, K., Wei, M.X., Harsh 4th, G.R., Tamiya, T &

Chiocca, E.A (1997) Regression of experimental brain tumors

with 6-thioxanthine and Escherichia coli gpt gene therapy Human

Gene Ther 8, 2043–2055.

30 Cheong, K.K., Fu, Y.Ch, Robins, R.K & Eyring, H (1969)

Absorption, optical rotatory dispersion, and circular dichroism

studies on some sulfur-containing ribonucleosides J Phys Chem.

73, 4218–4227.

31 Stoeckler, J.D., Poirot, A.F., Smith, R.M., Parks Jr, R.E., Ealick, S.E., Takabayashi, K & Erion, M.D (1997) Purine nucleoside phosphorylase 3 Reversal of purine base specificity by site-directed mutagenesis Biochemistry 36, 11749–11756.

32 Krenitsky, T.A., Elion, G.B., Henderson, A.M & Hitchings, G.H (1968) Inhibition of human purine nucleoside phosphorylase Studies with intact erythrocytes and the purified enzyme J Biol Chem 243, 4779–4784.

33 Mao, C., Cook, W.J., Zhou, M., Federov, A.A., Almo, S.C & Ealick, S.E (1998) Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues Biochemistry

37, 7135–7146.

34 Fedorov, A., Shi, W., Kicska, G., Fedorov, E., Tyler, P.C., Furneaux, R.H., Hanson, J.C., Gainsford, G.J., Larese, J.Z., Schramm, V.L & Almo, S.C (2001) Transition state structure of purine nucleoside phosphorylase and principles of atomic motion

in enzymatic catalysis Biochemistry 40, 853–860.

35 Ealick, S.E., Babu, Y.S., Bugg, C.E., Erion, M.D., Guida, W.C., Montgomery, J.A & Secrist 3rd, J.A (1991) Application of crystallographic and modeling methods in the design of purine nucleoside phosphorylase inhibitors Proc Natl Acad Sci USA

88, 11540–11544.

36 Koellner, G., Luic, M., Shugar, D., Saenger, W & Bzowska, A (1997) Crystal structure of calf spleen purine nucleoside phos-phorylase in a complex with hypoxanthine at 2.15 A˚ resolution.

J Mol Biol 265, 202–216.

37 Koellner, G., Luic, M., Shugar, D., Saenger, W & Bzowska, A (1998) Crystal structure of the ternary complex of E coli purine nucleoside phosphorylase with formycin B, a structural analogue

of the substrate inosine, and phosphate (Sulphate) at 2.1 A˚ resolution J Mol Biol 280, 153–166.

38 Erion, M.D., Takabayashi, K., Smith, H.B., Kessi, J., Wagner, S., Honger, S., Shames, S.L & Ealick, S.E (1997) Purine nucleoside phosphorylase 1 Structure–function studies Biochemistry 36, 11725–11734.

39 Erion, M.D., Stoeckler, J.D., Guida, W.C., Walter, R.L & Ealick, S.E (1997) Purine nucleoside phosphorylase 2 Catalytic mechanism Biochemistry 36, 11735–11748.

40 Montgomery, J.A (1993) Structure-based drug design: inhibitors

of purine nucleoside phosphorylase Farmaco 48, 297–308.

41 Farutin, V., Masterson, L., Andrcopulo, A.D., Cheng, J., Riley, B., Hakimi, R., Frazer, J.W & Cordes, E.H (1999) Structure– activity relationship for a class of inhibitors of purine nucleoside phosphorylase J Med Chem 42, 2422–2431.

42 Tebbe, J., Bzowska, A., Wielgus-Kutrowska, B., Schro¨der, W., Kazimierczuk, Z., Shugar, D., Saenger, W & Koellner, G (1999) Crystal structure of the purine nucleoside phosphorylase (PNP) from Cellulomonas sp and its implication for the mechanism of trimeric PNPs J Mol Biol 294, 1239–1255.

43 Montgomery, J.A., Niwas, S., Rose, J.D., Secrist, J.A., 3rd, Babu, Y.S., Bugg, C.E., Erion, M.D., Guida, W.C & Ealick, S.E (1993) Structure-based design of inhibitors of purine nucleoside phos-phorylase 1 9-(arylmethyl) derivatives of 9-deazaguanine J Med Chem 36, 55–69.

44 Franchetti, P & Grifantini, M (1999) Nucleoside and non-nucleoside IMP dehydrogenase inhibitors as antitumor and antiviral agents Curr Med Chem 6, 599–614.

45 Chung, J.H., Back, J.H., Park, Y.I & Han, Y.S (2001) Bio-chemical characterization of a novel hypoxanthine/xanthine dNTP pyrophosphatase from Methanococcus jannaschii Nucleic Acids Res 29, 3099–3107.

46 Ashihara, H & Crozier, A (1999) Biosynthesis and catabolism of caffeine in low-caffeine-containing species of Coffea J Agric Food Chem 47, 3425–3431.

47 Vos, S., Parry, R.J., Burns, M.R., de Jersey, J & Martin, J.L.

(1998) Structures of free and complexed forms of Escherichia coli

xanthine-guanine phosphoribosyltransferase J Mol Biol 282,

875–889.

48 Shivashankar, K., Subbayya, I.N & Balaram, H (2001)

Devel-opment of a bacterial screen for novel Hx-Gua PRTase substrates.

J Mol Microbiol Biotechnol 3, 557–562.

49 Vos, S., de Jersey, J & Martin, J.L (1997) Crystal structure of

Escherichia coli xanthine phosphoribosyltransferase Biochemistry

36, 4125–4134.

50 Craig 3rd, S.P & Eakin, A.E (2000) Purine

phosphoribosyl-transferases J Biol Chem 275, 20231–20234.

51 Jardim, A., Bergeson, S.E., Shih, S., Carter, N., Lucas, R.W.,

Merlin, G., Myler, P.J., Stuart, K & Ullman, B (1999) Xanthine

phosphoribosyltransferase from Leishmania donovani Molecular

cloning, biochemical characterization, and genetic analysis J Biol.

Chem 274, 34403–34410.

52 Kim, J.H., Ryan, M.G., Knaut, H & Hille, R (1996) The

reductive half-reaction of xanthine oxidase The involvement of

prototropic equilibria in the course of the catalytic sequence.

J Biol Chem 271, 6771–6780.

53 Sau, A.K., Mondal, M.S & Mitra, S (2000) Effects of pH and

temperature on the reaction of milk Xanthine oxidase with

1-methylxanthine J Chem Soc Dalton Trans 2000, 3688–3692.

54 Horlacher, J., Hottiger, M., Podust, V.M., Hubscher, U &

Benner, S.A (1995) Recognition by viral and cellular DNA

polymerases of nucleosides bearing bases with nonstandard

hydrogen bonding patterns Proc Natl Acad Sci USA 92,

6329–6333.

55 Vaish, N.K., Fraley, A.W., Szostak, J.W & McLaughlin, L.W.

(2000) Expanding the structural and functional diversity of RNA:

analog uridine triphosphates as candidates for in vitro selection of nucleic acids Nucleic Acids Res 28, 3316–3322.

56 Ohtsuki, T., Kimoto, M., Ishikaw a, M., Mitsui, T., Hirao, I & Yokoyama, S (2001) Unnatural base pairs for specific transcrip-tion Proc Natl Acad Sci USA 98, 9422–9425.

57 Fikus, M & Shugar, D (1969) Properties of poly-xanthylic acid and its reactions with potentially complementary homo-polynucleotides Acta Biochim Polon 16, 55–84.

58 Roy, K.B., Frazier, J & Miles, H.T (1979) Polixanthylic acid: Structures of the ordered forms Biopolymers 18, 3077–3087.

59 Arnott, S., Chandrasekaran, R., Day, A.W., Puigjaner, L.C & Watts, L (1981) Double-helical structures for polyxanthylic acid.

J Mol Biol 149, 489–505.

60 Pfleiderer, W & Nu¨bel, G (1961) Zur struktur des xanthins und seiner N-methyl-xanthin-derivate Ann Chem 647, 155–160.

61 Bzowska, A., Ananiev, A.V., Ramzaeva, N., Alksins, E., Maurins, J.A., Kulikowska, E & Shugar, D (1994) Purine nucleoside phosphorylase: inhibition by purine N(7)- and N(9)-acyclonu-cleosides; and substrate properties of 7-beta- D -ribofurano-sylguanine and 7-beta- D -ribofuranosylhypoxanthine Biochem Pharmacol 48, 937–947.

62 Porter, D.J (1992) Purine nucleoside phosphorylase Kinetic mechanism of the enzyme from calf spleen J Biol Chem 26, 7342–7351.

63 Wierzchowski, J., Wielgus-Kutrowska, B & Shugar, D (1996) Fluorescence emission properties of 8-azapurines and their nucleosides, and application to the kinetics of the reverse synthetic reaction of purine nucleoside phosphorylase Biochim Biophys Acta 1290, 9–17.