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.
Vertessy
B G
BG
Fachrichtung Biochemie, Universität des Saarlandes, Saarbrücken, Germany.
Zalud
P
P
Nyman
P O
PO
Zeppezauer
M
M
D000107:Acetylation; D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D007093:Imidazoles / Q000494:pharmacology; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine / Q000032:analysis*
D007093:Imidazoles; D014443:Tyrosine; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D013774:Tetranitromethane; C012160:N-acetylimidazole