Kinetic characterization of dUTPase from Escherichia coli.
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 Km for dCTP. With dUTP.Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s-1. Km 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 pKa values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP middle dotMg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-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 hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the alpha-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 dUTPase.UDP complex.
Larsson
G
G
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Lund, Sweden.
Nyman
P O
PO
Kvassman
J O
JO
D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D011755:Pyrophosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity
D003856:Deoxyuracil Nucleotides; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase; D008274:Magnesium