MODIFICATION WITH TETRANITROMETHANE OF AN ESSENTIAL TYROSINE RESIDUE IN URIDINE PHOSPHORYLASE FROM ESCHERICHIA-COLI

Treatment with tetranitromethane (TNM) rapidly and irreversibly inacti vates uridine phosphorylase (UPase) from E. coli under mildly alkaline conditions. Modification of one of the four tyrosine residues decreas es enzyme activity to 10%, while modification of all tyrosines decreas es it to 8%. The second-order rate constant for the inactivation is 12 50 +/- 50 M(-1) min(-1) at ph 8.0. Phosphate (0.1 m) does not affect t he inactivation rate, while 5 mM uridine, or uridine plus phosphate ne arly completely protect the enzyme against inactivation. Free sulfhydr yl groups of UPase are not oxidized by TNM. A single modified peptide was isolated from tryptic digest by reverse-phase HPLC. The mass to ch arge ratio and the sequence determined are consisted with modification of Tyr-169, which corresponds to tryptic peptide (169)Tyr-Asp-Thr-Tyr -Ser-Gly-Arg(175). Tyrosine nitration leads to a significant decrease in the pK(a) of the phenolic hydroxy group without significantly affec ting enzyme structure. Comparison of the pH dependence of activity and inactivation by diethylpyrocarbonate for the native and modified UPas e reveals interaction between the modified tyrosine residue and an ess ential histidine residue (Drabikowska, A.K. and Wozniak, G. (1990) Bio chem. J. 270, 319-323). It is suggested that Tyr-169 takes part in the stabilization of the imidazole ring of the essential histidine in UPa se.