Modification with tetranitromethane of an essential tyrosine residue in uridine phosphorylase from Escherichia coli.
Treatment with tetranitromethane (TNM) rapidly and irreversibly inactivates uridine phosphorylase (UPase) from E. coli under mildly alkaline conditions. Modification of one of the four tyrosine residues decreases enzyme activity to 10%, while modification of all tyrosines decreases it to 8%. The second-order rate constant for the inactivation is 1250 +/- 50 M-1 min-1 at pH 8.0. Phosphate (0.1 M) does not affect the inactivation rate, while 5 mM uridine, or uridine plus phosphate nearly completely protect the enzyme against inactivation. Free sulfhydryl groups of UPase are not oxidized by TNM. A single modified peptide was isolated from tryptic digest by reverse-phase HPLC. The mass to charge ratio and the sequence determined are consisted with modification of Tyr-169, which corresponds to tryptic peptide 169Tyr-Asp-Thr-Tyr-Ser-Gly-Arg175. Tyrosine nitration leads to a significant decrease in the pKa of the phenolic hydroxy group without significantly affecting enzyme structure. Comparison of the pH dependence of activity and inactivation by diethylpyrocarbonate for the native and modified UPase reveals interaction between the modified tyrosine residue and an essential histidine residue (Drabikowska, A.K. and Wozniak, G (1990) Biochem. J. 270, 319-323). It is suggested that Tyr-169 takes part in the stabilization of the imidazole ring of the essential histidine in UPase.
Komissarov
A A
AA
University of Missouri-Columbia, Department of Biochemistry 65212, USA.
Debabov
V G
VG
D000595:Amino Acid Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D013774:Tetranitromethane; D014357:Trypsin; D014443:Tyrosine / Q000737:chemistry*; D014529:Uridine / Q000378:metabolism; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism
D014443:Tyrosine; D014543:Uridine Phosphorylase; D014357:Trypsin; D013774:Tetranitromethane; D004047:Diethyl Pyrocarbonate; D014529:Uridine