title: Substrate specificity of E. coli uridine phosphorylase. Further evidences of high-syn conformation of the substrate in uridine phosphorolysis.
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.
authors.affiliation: a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , Moscow , Russia.
authors.forename: C S
authors.initials: CS
authors.lastname: Alexeev
authors.affiliation: b Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus , Minsk , Belarus.
authors.forename: G G
authors.initials: GG
authors.lastname: Sivets
authors.affiliation: c Bach Institute of Biochemistry, Russian Academy of Sciences , Moscow , Russia.
authors.forename: T N
authors.initials: TN
authors.lastname: Safonova
authors.affiliation: a Engelhardt Institute of Molecular Biology, Russian Academy of Sciences , Moscow , Russia.
authors.forename: S N
authors.initials: SN
authors.lastname: Mikhailov
mesh_terms: D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D013379:Substrate Specificity; D014529:Uridine / Q000737:chemistry* / Q000378:metabolism*; D014543:Uridine Phosphorylase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D014543:Uridine Phosphorylase; D014529:Uridine
keywords: Uridine phosphorylase; conformation of uridine; modified uridines; substrate specificity