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Enzyme‐catalyzed uridine phosphorolysis: S N 2 mechanism with phosphate activation by desolvation
Author(s) -
Komissarov A.A.,
Moltchan O.K.,
Romanova D.V.,
Debabov V.G.
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)01204-0
Subject(s) - phosphorolysis , chemistry , uridine , fluorescein , substrate (aquarium) , active site , enzyme , stereochemistry , biochemistry , purine nucleoside phosphorylase , fluorescence , rna , physics , oceanography , quantum mechanics , purine , gene , geology
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 S N 2 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 S N 2 uridine phosphorolysis reaction.