KINETIC CHARACTERIZATION OF DUTPASE FROM ESCHERICHIA-COLI

The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyroph osphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, lik e the human enzyme, consists of three identical subunits. In the prese nt work, the catalytic properties of the E. coli dUTPase were investig ated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphat e moiety (bound dUDP was not hydrolyzed). The second best substrate am ong the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10(5) times less efficiently than dUTP, a decline largely accounted for by a higher K-m for dCTP. With dUTP M g as substrate, k(cat) was found to vary little with pH and to range f rom 6 to 9 s(-1). K-m passed through a broad minimum in the neutral pH range with values approaching 10(-7) M. It increased with deprotonati on of the uracil moiety of dUTP and showed dependence on two ionizatio ns in the enzyme, exhibiting pK(a) values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP Mg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the ra te-limiting step, The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2'-deoxyuridine-5'-(alpha-thio)-triphosphate was hyd rolyzed by the enzyme. These results are interpreted to favor a cataly tic mechanism involving magnesium binding to the cy-phosphate, rate-li miting hydrolysis by a shielded and activated water molecule and a fas t ordered desorption of the products. The results are discussed with r eference to recent data on the structure of the E. coli dUTPase dUDP c omplex.