Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from Hela S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from Hela S3 cells appears to be composed of two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl₂. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl₂ will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl₂ will also promote association of the dUTPase subunits. However, MnCl₂ will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl₂. In contrast, addition of 5 mM MnCl₂ to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg²⁺ is the natural prosthetic group for this enzyme. The K(m) value of dUTP for the purified enzyme is 3 x 10^-6 M in the presence of MgCl₂. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infections of Hela S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl₂. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.