Substrate specificity of thymidine phosphorylase of E. coli: Role of hydroxyl groups

Article abstract of DOI:10.1080/15257770802257895

Substrate specificity of E. coli thymidine phosphorylase to pyrimidine nucleoside modified at 5′-, 3′-, and 2′-positions of sugar moiety has been studied. Equilibrium (Keq) and kinetics constants of phosphorolysis reaction of nucleosides were measured. Th

Full text of DOI:10.1080/15257770802257895

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Substrate Specificity of Thymidine
Phosphorylase of E. Coli: Role of
Hydroxyl Groups
a
a
a
Natalya G. Panova , Cyril S. Alexeev , Konstantin M. Polyakov
,
a
a
a
Sergei A. Gavryushov , Anatoliy M. Kritzyn & Sergey N. Mikhailov
a
Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences , Moscow, Russia
Published online: 26 Nov 2008.
To cite this article: Natalya G. Panova , Cyril S. Alexeev , Konstantin M. Polyakov , Sergei A.
Gavryushov , Anatoliy M. Kritzyn & Sergey N. Mikhailov (2008) Substrate Specificity of Thymidine
Phosphorylase of E. Coli: Role of Hydroxyl Groups, Nucleosides, Nucleotides and Nucleic Acids, 27:12,
1211-1214, DOI: 10.1080/15257770802257895
To link to this article: http://dx.doi.org/10.1080/15257770802257895
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Nucleosides, Nucleotides and Nucleic Acids, 27:1211–1214, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770802257895
SUBSTRATE SPECIFICITY OF THYMIDINE PHOSPHORYLASE
OF E. COLI: ROLE OF HYDROXYL GROUPS
Natalya G. Panova, Cyril S. Alexeev, Konstantin M. Polyakov,
Sergei A. Gavryushov, Anatoliy M. Kritzyn, and Sergey N. Mikhailov
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
ꢁ
2
Substrate specificity of E. coli thymidine phosphorylase to pyrimidine nucleoside modified at 5 -,
ꢁ
ꢁ
3
-, and 2 -positions of sugar moiety has been studied. Equilibrium (Keq) and kinetics constants
of phosphorolysis reaction of nucleosides were measured. The most important hydrogen bonds in
enzyme-substrate complex have been determined.
Keywords Thymidine phosphorlase; substrate specificity; thymidine derivatives
INTRODUCTION
Thymidine phosphorylase (TP) belongs to a class of nucleoside phos-
phorylases, which are involved in the processes of nucleoside methabolism
ꢁ
in cell. It catalyzes the reversible reaction of phosphorolysis of 2 -
ꢁ
deoxypyrimidines to heterocyclic base and 2 -deoxy-α-D-ribose-1-phosphate
[
1,2]
and normally the equilibrium shifts to nucleoside:
dRib-P.
Thd + Pi ↔ Thy +
Thus TP participates in the salvage way of nucleoside synthesis and main-
tains the thymidine pool in cell. TPs were found in all live organisms and
[
1]
it appeared that their homology is nearly 40%.
The three-dimensional
structures of TPs from different sources were determined and a key role for
highly conserved amino acids and their hydrogen bonds with pyrimidine
base was found.[ On the bases of the structure of bacterial TP and molec-
ular dynamic simulations it has been shown that substrate binding starts a
3]
Received 2 November 2006; accepted 27 March 2008.
This manuscript was presented at the 2006 Round Table Conference, XVII International
Roundtable, Bern, Switzerland, September 3–7, 2006.
Financial support of the Russian Foundation for Basic Research is acknowledged.
Address correspondence to Sergey N. Mikhailov, Engelhardt Institute of Molecular Biology, Russian
Academy of Sciences, Vavilov Street 32, Moscow 119991, Russia. E-mail: smikh@eimb.ru
1
211

1
212
N. G. Panova et al.
domain movement, which leads to the “closed” conformation of the active
[
4]
site and initiates phosphorolysis. The level of nucleoside phosphorylases
[
5]
is higher in numerous cancers than in normal cells. Efficient inhibitors
of these enzymes are promising as potential anticancer drugs, suitable not
only for direct treatment of cancer, but also for prolonging the effect of
other drugs.
RESULTS AND DISCUSSION
Nucleoside phosphorylases are widely used in industry for synthesis of
[
6,7]
drugs and commercially important nucleosides.
The aim of this work
was to study substrate and inhibitory properties of thymidine derivatives in
[
8]
the reactions catalyzed by recombinant E. coli TP.
Substrate properties
of a series of sugar modified nucleosides were investigated (Figure 1).
ꢁ
5
-Deoxythymidine (2) showed good substrate properties according to its
ꢁ
kinetic constants, while replacement of the 5 -OH with bulky substituents
(
3–6) or an amino group (7), or the introduction of methyl (8, 9) or
methylene (10) groups did not hinder binding but led to a significant
reduction in the reaction rate (Table 1).
ꢁ
Modification of the 3 -OH dramatically influenced the binding of nu-
cleosides to TP. Nucleosides 11–13 were not substrates but inhibited TP
activity with a K close to the K of the natural substrate. On the bases of
I
M
the “closed structure” of active site (model structure was obtained using
molecular dynamic simulation)[ we concluded that the 3 -hydroxyl group
9]
ꢁ
a
TABLE 1 Kinetic constants of modified nucleosides in phosphorolysis reaction catalyzed by TP
KM(KI) · 10−
6
M
−1
kcat, s
kcat/KM
Nucleoside
Keq(1/Keq)
1
2
3
4
5
6
7
8
9
0.07(14.3)
0.05 (20)
0.07(14.3)
300
400
300
(400)
(400)
(>1000)
400
330
350
300
198
260
1.7
0.66
0.64
0.006
0.58
0.1
0.45
0.6
0.0015
0.0003
0.0013
0.002
0.14 (7)
0.10 (10)
0.068(14.6)
10
11
12
13
14
(850)
(600)
(450)
350
ND
0.4
0.0011
a
The kinetic parameters for phosphorolysis of thymidine analogs in the presence of TP were
[
8]
determined spectrophotometrically.
and 37 C.
The equilibrium constants were measure using HPLC at pH 6.5
◦
[8]

Substrate Specificity of Thymidine Phosphorylase of E. coli
1213
FIGURE 1 Structure of thymidine derivatives.
formed a hydrogen bond with Thr123 and due to this bond nucleoside was
moved into a position suitable for nucleophilic attack by phosphate.
ꢁ
In conclusion, the absence of a 5 -hydroxyl group leads to no significant
differences in binding constants and reaction rate, but introduction of
bulky groups can hinder the domain movement because of their close
proximity to the Gly88 residue, thereby lessening the reaction rate, while
ꢁ
the 3 -hydroxyl group is essential for phosphorolysis reaction.
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