Kinetic Characterization of dUTPase from Escherichia coli

Abstract

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...

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

“…The reaction is initiated by the attack of the nucleophilic water, leading to an associative pathway for the phosphate cleavage that has been identified in solution and in several enzymes that carry out phosphate cleavage or transfer (see e.g., Florián et al [53][54] and Klahn et al 55 ). This mechanism is also consistent with the experimentally observed pH change that is used to directly follow the reaction kinetics, 56 and with the lack of observed intermediates for the phosphate hydrolysis. 33 It is found in E. coli dUTPase that two protons transfer to the reaction medium in a concerted mode, immediately following the rate limiting step.…”

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

“…This conformational change is realized by intricate subunit interactions as the C terminus of one subunit crosses over its neighboring subunit and adopts an ordered conformation to contact the substrate bound in the active site of the third subunit. In the bacterial enzyme, detailed functional investigations (2,30,33,34), in agreement with the crystal structure in complex with the nucleotide dUDP (28), indicate that the ␥ phosphate of the substrate is indispensable in inducing the closed conformer, and after catalytic cleavage the active site pops open. In the case of the human enzyme, the crystal structure suggests the retaining of the closed conformer even with the product dUMP (24), but there are no functional data published.…”

“…Experiments to identify the physiological isoforms of fly dUTPase are in progress in our laboratory. 2 Both the N and C termini of fly dUTPase may contain subcellular localization segments sensitive to proteolysis (Fig. 5), arguing for their exposed character and accessibility that might be helpful in binding to potential transport proteins.…”

“…This method was used to conveniently follow activity during purification and trypsinolysis, as well as to record substrate saturation curves with the dUTP analogues dCTP and dTTP. The enzyme kinetic parameters k cat and K M were determined either from substrate saturation curves or from the entire progress curve of dUTP cleavage using the integrated Michaelis-Menten equation, as described previously (2,33). Metal ion requirement was tested in separate experiments, using Chelex-treated components.…”

“…The enzyme dUTPase catalyzes the hydrolysis of dUTP into dUMP and anorganic pyrophosphate using a metal ion (Mg 2ϩ ) (1,2). Through this reaction, the enzyme keeps the cellular dUTP/dTTP ratio at a low level by depleting the dUTP pool and contributing in parallel to dTTP biosynthesis (3).…”