title: Enzyme-catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation.
abstract: The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.
authors.affiliation: State Scientific Centre of Russian Federation GNIIGENETIKA, Moscow.
authors.forename: A A
authors.initials: AA
authors.lastname: Komissarov
authors.forename: O K
authors.initials: OK
authors.lastname: Moltchan
authors.forename: D V
authors.initials: DV
authors.lastname: Romanova
authors.forename: V G
authors.initials: VG
authors.lastname: Debabov
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D016650:Fluorescein-5-isothiocyanate; D007700:Kinetics; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism; D012997:Solvents; D014529:Uridine / Q000737:chemistry / Q000378:metabolism*; D014543:Uridine Phosphorylase / Q000378:metabolism
chemical_list: D010710:Phosphates; D012997:Solvents; D014543:Uridine Phosphorylase; D016650:Fluorescein-5-isothiocyanate; D014529:Uridine