ENZYME-CATALYZED URIDINE PHOSPHOROLYSIS - S(N)2 MECHANISM WITH PHOSPHATE ACTIVATION BY DESOLVATION

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In con trast, the rate was increased about twofold after preincubation of uri dine phosphorylase with 60% acetonitrile. These data correlate with kn own effects of polar and bipolar aprotic solvents on S(N)2 nucleophili c substitution reactions. The enzyme modified with fluorescein-5'-isot hiocyanate (fluorescein residue occupies an uridine-binding subsite [K omissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was sele ctively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphoryl ase is assumed to imitate the enzyme-substrate complex. Modification w ith SA-423 was accompanied with dramatic changes in the absorption spe ctrum 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 uri dine phosphorolysis reaction.