Identification of tyrosine as a functional residue in the active site of Escherichia coli dUTPase.

dUTP nucleotidohydrolase (dUTPase, EC 3.6.1.23) from E. coli contains a total of six tyrosine residues per trimer. About half of them were found to be susceptible to acetylation with N-acetylimidazole or to nitration with tetranitromethane with concomitant loss of activity. Deacetylation with N-hydroxylamine leads to full reactivation. Inhibitory products of dUTP hydrolysis, i.e., dUMP and inorganic pyrophosphate together with the cofactor Mg2+ protect significantly against inactivation and chemical modification. In the Cu(2+)-dUTPase complex, charge transfer from Cu2+ to the tyrosinate anion was perturbed by the presence of the substrate dUTP. These results, together with the occurrence of one tyrosine residue in a strictly conserved sequence motif suggest the critical importance of this residue for the function of the enzyme.