Kinetic properties of Cellulomonas sp. purine nucleoside phosphorylase with typical and non-typical substrates: Implications for the reaction mechanism

Article abstract of DOI:10.1081/NCN-200060011

Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino), and non-typical 7-methylguanosine (m⁷Guo), with either nucleoside or phosphate (P_i) as the varied substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP, complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of me substrate purine bases, leading to the circumstances that for such substrates (products) rapid-equilibrium assumptions do not hold, and a dual role of P_i, a substrate, and also a reaction modifier that helps to release a tightly bound purine base. Copyright Taylor & Francis, Inc.

Full text of DOI:10.1081/NCN-200060011

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KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE
NUCLEOSIDE PHOSPHORYLASE WITH TYPICAL AND
NON-TYPICAL SUBSTRATES: IMPLICATIONS FOR THE
REACTION MECHANISM
Beata Wielgus-Kutrowska a & Agnieszka Bzowska a
a Department of Biophysics , Institute of Experimental Physics, University of Warsaw ,
Warsaw, Poland
Published online: 15 Nov 2011.
To cite this article: Beata Wielgus-Kutrowska & Agnieszka Bzowska (2005) KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE
NUCLEOSIDE PHOSPHORYLASE WITH TYPICAL AND NON-TYPICAL SUBSTRATES: IMPLICATIONS FOR THE REACTION MECHANISM,
Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 471-476, DOI: 10.1081/NCN-200060011
To link to this article: http://dx.doi.org/10.1081/NCN-200060011
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):471–476, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200060011
KINETIC PROPERTIES OF CELLULOMONAS SP. PURINE NUCLEOSIDE
PHOSPHORYLASE WITH TYPICAL AND NON-TYPICAL SUBSTRATES:
IMPLICATIONS FOR THE REACTION MECHANISM
5
Beata Wielgus-Kutrowska and Agnieszka Bzowska
Department of Biophysics,
Institute of Experimental Physics, University of Warsaw, Warsaw, Poland
5
Phosphorolysis catalyzed by Cellulomonas sp. PNP with typical nucleoside substrate, inosine (Ino),
and non-typical 7-methylguanosine (m7Guo), with either nucleoside or phosphate (Pi) as the varied
substrate, kinetics of the reverse synthetic reaction with guanine (Gua) and ribose-1-phosphate (R1P) as
the varied substrates, and product inhibition patterns of synthetic and phosphorolytic reaction pathways
were studied by steady-state kinetic methods. It is concluded that, like for mammalian trimeric PNP,
complex kinetic characteristics observed for Cellulomonas enzyme results from simultaneous occurrence
of three phenomena. These are sequential but random, not ordered binding of substrates, tight binding of
one substrate purine bases, leading to the circumstances that for such substrates (products) rapid-
equilibrium assumptions do not hold, and a dual role of Pi , a substrate, and also a reaction modifier
that helps to release a tightly bound purine base.
Keywords Purine Nucleoside Phosphorylase, Catalytic Mechanism, Enzyme Kinetics,
Non-Michaelis-Menten Kinetics
INTRODUCTION
Purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) is a ubiquitous enzyme of
the purine salvage metabolic pathway. In eukaryotes and in some bacteria trimeric
enzyme is present which catalyses the reversible phosphorolysis of 6-oxo-purine
ribo- and 2’-deoxyribonucleosides, as follows: b-D-purine nucleoside + orthophos-
phate () purine base + a-D-pentose-1-phosphate. Hexameric PNP of prokaryotic
origin accepts also 6-amino-purine and their nucleosides as substrates. Both classes
of PNPs are important targets for chemotherapy. Potent inhibitors of human and
parasitic PNPs are considered potential immunosuppressive and antiparasitic
agents, respectively, while broad specificity of some prokaryotic phosphorylases
Supported by Polish State Committee for Scientific Research KBN (grant 3P04A 035 24).
Address correspondence to Agnieszka Bzowska, Department of Biophysics, Institute of Experimental Physics,
University of Warsaw, 93 Zwirki & Wigury St., Warsaw 02-089, Poland; E-mail: abzowska@biogeo.uw.edu.pl
471
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B. Wielgus-Kutrowska and A. Bzowska
makes them ideal candidates for gene therapy of some tumours and also a tool for
enzymatic synthesis of nucleosides.[1]
The mechanism of action of PNP still needs to be clarified and one of the
important questions regards possible interactions of enzyme subunits upon
substrate binding and in the process of catalysis. PNPs isolated from various
sources show complex kinetic characteristics which could indicate cooperativity
between monomers forming the entire enzyme.[1] In fact, for hexameric E. coli PNP
it was demonstrated by X-ray studies in the crystal and fluorescence studies in
solution that binding of phosphate and nucleoside is associated with negative
cooperativity.[2,3] By contrast, for trimeric mammalian PNPs negative cooperativity
was documented only for transition-state events.[4–6] Ground-state analogues seem
to bind uniformly to all three sites of calf spleen PNP[7,8] and non-Michaelis kinetics
observed for some substrates seems to be the result of complex kinetic mechanism
and not of the cooperativity between enzyme subunits.[7] Here we extend our
studies in solution for trimeric, bacterial origin PNP (isolated from Cellulomonas sp.),
and show by kinetic methods that the above conclusion is valid not only for
mammalian, but also for bacterial trimeric PNP.
MATERIALS AND METHODS
Materials
Hepes (ultra pure), Na2HPO4, NaH2PO4, a-D-ribose-1-phosphate (R1P), inosine
(Ino), guanosine (Guo), 7-methylguanosine (m7Guo), guanine (Gua), hypoxanthine
(Hx), and other chemicals were products of Sigma or Fluka. All solutions were
prepared with high-quality MiliQ water. Commercially available PNP from
Cellulomonas sp. (Toyobo Co., Japan) was purified on a BioCad station (PerSeptive
Biosystems) by ion-exchange chromatography as described[9] to a maximal final
specific activity of 125 U/mg. Xanthine oxidase (XO) from buttermilk (1 U/mg at
25°C) was purchased from Sigma.
Concentrations
Concentration of Gua, nucleosides and PNP were determined spectrophoto-
metrically from their molar extinction coefficients at pH 7.0: Gua e246 = 10 700 MÀ1
cmÀ1, Ino e249 = 12,300 MÀ1cmÀ1, m7Guo e260 = 8,500 MÀ1cmÀ1, Guo
e252,5 = 13,650 MÀ1cmÀ1; PNP e280 = 9.6 cmÀ1 for a 1% solution.[7] The con-
centrations of ligands that show no absorbance in the UV-VIS region (phosphate, Pi,
and R1P) were calculated from their molecular mass.
Apparatus
Spectral data were collected on a Kontron (Switzerland) Uvikon 930. A
¨
Beckman model O300 pH-meter, was used for pH determinations. Data were
analyzed using the PC GraphPad Prism 3.0 (GraphPad Software Inc.) and Origin
6.0 (Microcal) computer software packages.

B. Wielgus-Kutrowska and A. Bzowska
Kinetic Procedures
473
The specific activity of PNP was measured spectrophotometrically with the use
of Kalckar’s method, where inosine was the substrate and the product,
hypoxanthine (Hx), was oxidized by xanthine oxidase to form uric acid,[10]
De = 9,600 MÀ1cmÀ1 for 300 nm. With Guo and m7Guo as substrates a direct assay
was employed based on marked differences in extinction coefficients of these
nucleosides and products of their phosphorolysis: De = 4,600 MÀ1cmÀ1 at 260 nm
at pH 7.0 for m7Guo and De = 5,500 MÀ1cmÀ1 at 258 nm for Guo.[7]
The reaction mixtures contained 50 mM Hepes pH 7.0, both substrates
(nucleoside and phosphate for the measurements of the phosphorolytic reaction, or
purine base and ribose-1-phosphate for the measurements of the synthetic reaction).
In the case of Ino phosphorolysis coupled assay was used, so xanthine oxidase was
also present, while for product inhibition studies the appropriate product was
included in the reaction mixture. Cuvettes with 10 mm, 2 mm, or 1 mm path length
were used. The reaction was started by the addition of PNP. The initial velocity of
the reactions was calculated by the linear regression of absorbance versus time.
Lineweaver-Burk transformation was used to analyze kinetics of phosphorolysis
and synthesis, and product inhibition data.[11] Eqs. 1 and 2 were fitted to kinetic
data of phosphorolysis obtained over a broad concentration range of a variable
substrate. Statistically significant decrease in the sum of residuals, judged by the
Snedecor’s F test at a 95% confidence level, residual plots, and P value from the runs
test were used to determine whether the data differ significantly from the equation
selected and to discriminate between possible models.
RESULTS
Kinetics of phosphorolysis and synthesis for Ino, Pi, R1P, and Gua were
measured for a variable concentration of one substrate and for four different,
constant concentrations of a second substrate. For these conditions linear double
reciprocal plots were obtained (but see below). The crossing of straight lines
observed for phosphorolysis indicate the sequential mechanism, while the paral-
lelism of lines for synthesis surprisingly suggest the ping-pong mechanism for this
reaction direction.[11]
To verify these contradictory results product inhibition pattern and apparent
inhibition constants were determined for the phosphorolytic and synthetic reactions
with Ino and Pi, and Gua and R1P as the varied substrates and respective products.
Gua was a competitive inhibitor for Pi and for Ino as the varied substrates, with Ki
in mM range (2.5 0.5 mM and 3.6 0.6 mM, respectively). R1P causes mixed
inhibition, with Ki in mM range for Pi (25 5 mM; 320 40 mM) and in mM range
for Ino (7.5 0.5 mM; 10.2 0.4 mM) as the varied substrates. The synthesis
reaction was inhibited much weaker by the products (Guo and Pi), either with Ki in
mM range or, in one case, no inhibition was observed (for Gua as the varied
substrate and Guo as the inhibitor). Inhibition of R1P by Guo (Ki ꢀ0.8 mM), and

474
B. Wielgus-Kutrowska and A. Bzowska
Gua by Pi (Ki 4.7 1.0 mM) is uncompetitive, while of R1P by Pi is competitive
(Ki = 2.5 0.8 mM). The inhibition pattern described above does not agree with
any Bi–Bi reaction mechanism.[12]
In the next step the phosphorolytic reaction of inosine and 7-methylguanosine
was investigated over a broad concentration range of a varied substrate (Pi,
Figure 1, and nucleoside, not shown), and, in the case of Ino as a varied substrate,
at various levels of a co-substrate. Kinetics (initial velocity, vo versus variable
substrate concentration, co) is clearly non-hyperbolic for some substrates. Kinetic
data was therefore analyzed either by the rational function of degree 1:1 (Michaelis-
Menten equation):
Vmaxco
noðcoÞ ¼
ð1Þ
co þ Km
or by the rational function of degree 2:2, which is the mathematical expression
describing several reaction mechanisms (interpretation of constants i, j, l, and m
depends on the mechanism):[11]
jco þ ic2o
noðcoÞ ¼
ð2Þ
1 þ mco þ lc2o
The Michaelis-Menten model is sufficient for phosphate (Pi) as the varied
substrate with 200 mM of m7Gua (Figure 1, panel B) and for Ino as the varied
substrate with 0.5 mM and 50 mM of Pi (not shown). However, for the Pi as the
FIGURE 1 Kinetics of phosphorolysis of Ino (A) and m7Guo (B) studied with Pi as variable substrate. Reactions
were conducted at 25°C in 50 mM Hepes buffer pH 7.0 and followed by the coupled XOassay for Ino[10] and
direct assay for m7Guo.[15] In both cases, Michaelis-Menten equation and rational function of degree 2:2 were fitted
(solid and broken lines, respectively, see Eqs. 1 and 2). Residuals for both fits are shown as closed and open
symbols for Michaelis-Menten and 2:2 equation, respectively.

B. Wielgus-Kutrowska and A. Bzowska
475
varied substrate with 1 mM Ino (Figure 1, panel A), for Ino as the varied substrate
with 0.05 mM Pi, and for m7Guo with 50 mM Pi (not shown), the kinetic data could
only be described by the rational function of degree 2:2.
DISCUSSION
All experiments described above indicate that the reaction catalyzed by
Cellulomonas PNP, similar like the catalytic process for mammalian trimeric PNPs,[7]
cannot be sufficiently described by the Michaelis-Menten kinetic model. However,
deviations from this model are pronounced stronger or weaker and vary markedly
for various substrates. Hence, interpretation of kinetic data on the basis of Michaelis-
Menten equation is in principle incorrect and may lead to puzzling and
contradictory conclusions like those presented in the first two paragraphs of the
Results. The kinetic data for Cellulomonas PNP could only be described by the
rational function of degree 2:2., i.e., by the model with quadratic terms in co, which is
characteristic for several reaction mechanisms, e.g., allosteric interactions between
enzyme subunits but also steady-state random systems.[11] Steady-state kinetic data
alone can not distinguish between these two mechanisms. However, data presented
here, together with our earlier results[13,14] obtained by means of fluorimetric
titrations and protective effects of ligands on the thermal inactivation of the enzyme
point to the steady-state random system. We have shown: 1) formation of binary
complexes with the enzyme for all substrates (purine base, R1P, Pi, and nucleoside),
2) stoichiometry of three molecules bound per enzyme trimer for several ground-
state ligands, and 3) potent binding of some purines like Gua and Hx, (but not
7-methylguanine), in the absence of Pi, and less potent binding when Pi is
present.[13,14] Therefore, it seems that for Cellulomonas sp. PNP, like for mammalian
trimeric PNPs,[7] complex kinetic characteristics results from simultaneous
occurrence of three phenomena. These are: 1) sequential but random, not ordered
binding of substrates, 2) tight binding of one substrate purine bases, e.g., Gua and
Hx, leading to the circumstances that for such substrates (products) rapid-
equilibrium assumptions do not hold, and 3) a dual role of Pi—a substrate and
also a reaction modifier that helps to release a tightly bound purine base. Under
these assumptions and limitations all puzzling results can be interpreted.
For example the rational function of degree 2:2 in general should be used to
describe kinetic data obtained for PNP from Cellulomonas sp. However, in some
cases when 1) either one substrate is present in saturating concentration and
randomness of the reaction disappears (phosphorolysis of Ino with 50 mM and
0.5 mM Pi) or 2) binding of product is not potent (e.g., for 7-methylguanine)
and rapid-equilibrium assumptions hold, kinetics simplifies to the one described by
the Michaelis-Menten equation.
Also the occurrence of the seemingly parallel lines for synthesis of Gua
pointing to the ping-pong mechanism, which for sure is not valid for PNP,[1] is
intelligible. Some sequential systems are known to appear as to be ping-pong,

476
B. Wielgus-Kutrowska and A. Bzowska
among others a non-rapid equilibrium random system where the rate constants for
release of substrates are lower than the maximal velocity.[11] In fact, release of Gua
is very slow especially when there is no phosphate, which helps in this process.
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