SELECTIVE MODIFICATION OF PUTATIVE URIDINE-BINDING SITE OF URIDINE PHOSPHORYLASE FROM ESCHERICHIA-COLI WITH FLUORESCEIN 5'-ISOTHIOCYANATE

A Putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5'-isothiocyanate (FITC). T reatment with FITC irreversibly inactivates the enzyme (K(i) = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorp oration of the reagent reaches about 1 mol per mol of the enzyme subun it. Addition of uridine prevents the enzyme inactivation by FITC. In c ontrast to this, addition of a second substrate phosphate increases th e rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effec t on the affinity of the reagent to the enzyme. The modified protein r etains the ability to bind phosphate but not uridine. According to dif ferential absorption spectroscopy data, the binding of phosphate to th e active site of the enzyme is accompanied by conformational changes w hich may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, whi le the phosphate-binding site still retains the ability to interact wi th the second substrate.