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The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated 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 phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among 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 dUTP⅐Mg as substrate, k cat was found to vary little with pH and to range from 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 deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pK a values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP⅐Mg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg 2؉ 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-(␣-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the ␣-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase⅐dUDP complex.The enzyme dUTPase (EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is an important factor for the prevention of uracil incorporation into DNA (1). Along with the reduced nucleotides required for the synthesis and repair of DNA, the ribonucleotide reductase converts UDP to dUDP. The latter becomes phosphorylated to dUTP, which is readily mistaken for dTTP by DNA polymerase and, hence, may become incorporated into DNA (2). The absence of dUTPase triggers an elevated recombination frequency and an abnormal mutation rate and leads to the accumulation of short DNA fragments as intermediates in the DNA metabolism (3). The adverse effects of a low dUTPase activity suggest that inhibitors of this enzyme may find a role as cytostatic drugs in cancer therapy (4). dUTPase is widespread in nature and has been found in a variety of eukaryotic and prokaryotic organisms as well as in many viruses (5-8). It has been proven to be essential for the viability of Escherichia coli (9) and Saccharomyces cerevisiae (10). In some organisms, a second major role for dUTPase is to provide dUMP for the synthesis of dTTP (11).The three-dimensional structure of dUTPase from E. coli has been determined to 1.9-Å resolution (12) and re...

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The enzyme dUTPase catalyses the hydrolysis of dUTP and maintains a low intracellular concentration of dUTP so that uracil cannot be incorporated into DNA. dUTPase from Escherichia coli is strictly specific for its dUTP substrate, the active site discriminating between nucleotides with respect to the sugar moiety as well as the pyrimidine base. Here we report the three-dimensional structure of E. coli dUTPase determined by X-ray crystallography at a resolution of 1.9 A. The enzyme is a symmetrical trimer, and of the 152 amino acid residues in the subunit, the first 136 are visible in the crystal structure. The tertiary structure resembles a jelly-roll fold and does not show the 'classical' nucleotide-binding domain. In the quaternary structure there is a complex interaction between the subunits that may be important in catalysis. This possibility is supported by the location of conserved elements in the sequence.

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