title: Selective modification of putative uridine-binding site of uridine phosphorylase from E. coli with fluorescein 5'-isothiocyanate.
abstract: A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5'-isothiocyanate (FITC). Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit. Addition of uridine prevents the enzyme inactivation by FITC. In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme. The modified protein retains the ability to bind phosphate but not uridine. According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.
authors.affiliation: Institute for Genetics of Microorganisms, Moscow, Russia.
authors.forename: A A
authors.initials: AA
authors.lastname: Komissarov
authors.forename: D V
authors.initials: DV
authors.lastname: Romanova
authors.forename: N A
authors.initials: NA
authors.lastname: Dmitrieva
authors.forename: E V
authors.initials: EV
authors.lastname: Linkova
authors.forename: A S
authors.initials: AS
authors.lastname: Mironov
authors.forename: V G
authors.initials: VG
authors.lastname: Debabov
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010710:Phosphates / Q000378:metabolism; D013053:Spectrophotometry; D014529:Uridine / Q000378:metabolism; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors*
chemical_list: D010710:Phosphates; D014543:Uridine Phosphorylase; D016650:Fluorescein-5-isothiocyanate; D014529:Uridine