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ABSTRACT
Twenty five uridine analogues have been tested and compared with uridine with respect to their potency to bind to E. coli uridine phosphorylase. The kinetic constants of the phosphorolysis reaction of uridine derivatives modified at 2′-, 3′- and 5′-positions of the sugar moiety and 2-, 4-, 5- and 6-positions of the heterocyclic base were determined. The absence of the 2′- or 5′-hydroxyl group is not crucial for the successful binding and phosphorolysis. On the other hand, the absence of both the 2′- and 5′-hydroxyl groups leads to the loss of substrate binding to the enzyme. The same effect was observed when the 3′-hydroxyl group is absent, thus underlining the key role of this group. Our data shed some light on the mechanism of ribo- and 2′-deoxyribonucleoside discrimination by E. coli uridine phosphorylase and E. coli thymidine phosphorylase. A comparison of the kinetic results obtained in the present study with the available X-ray structures and analysis of hydrogen bonding in the enzyme-substrate complex demonstrates that uridine adopts an unusual high-syn conformation in the active site of uridine phosphorylase.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
Author contributions
Conceived and designed the experiments: S.N. Mikhailov. Performed the kinetic experiments: C.S. Alexeev. Analyzed the data: S.N. Mikhailov. Analysis of crystallographic data: T. N. Safonova. Synthesis of 3′-deoxythymidine and 3′-fluoro-3′-deoxyuridine: G.G.Sivets. Wrote the paper: S.N. Mikhailov.
Acknowledgments
The research was supported by the Russian Science Foundation, grant No 16-14-00178. T. N. Safonova was supported by the Russian Science Foundation (RSF; grant No. 14-24-00172).
The authors are grateful to Dr. K.M. Polyakov and Dr. N.G. Panova for help and fruitful discussions.