Kinetic Characterization of dUTPase from Escherichia coli

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Kinetic Characterization of dUTPase from Escherichia coli. / Larsson, Gunilla; Nyman, Per-Olof; Kvassman, Jan-Olov.

In: Journal of Biological Chemistry, Vol. 271, No. 39, 1996, p. 24010-24016.

Research output: Contribution to journalArticle

Harvard

Larsson, G, Nyman, P-O & Kvassman, J-O 1996, 'Kinetic Characterization of dUTPase from Escherichia coli', Journal of Biological Chemistry, vol. 271, no. 39, pp. 24010-24016. <http://www.jbc.org/cgi/reprint/271/39/24010>

APA

CBE

Larsson G, Nyman P-O, Kvassman J-O. 1996. Kinetic Characterization of dUTPase from Escherichia coli. Journal of Biological Chemistry. 271(39):24010-24016.

MLA

Larsson, Gunilla, Per-Olof Nyman and Jan-Olov Kvassman. "Kinetic Characterization of dUTPase from Escherichia coli". Journal of Biological Chemistry. 1996, 271(39). 24010-24016.

Vancouver

Larsson G, Nyman P-O, Kvassman J-O. Kinetic Characterization of dUTPase from Escherichia coli. Journal of Biological Chemistry. 1996;271(39):24010-24016.

Author

Larsson, Gunilla ; Nyman, Per-Olof ; Kvassman, Jan-Olov. / Kinetic Characterization of dUTPase from Escherichia coli. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 39. pp. 24010-24016.

RIS

TY - JOUR

T1 - Kinetic Characterization of dUTPase from Escherichia coli

AU - Larsson, Gunilla

AU - Nyman, Per-Olof

AU - Kvassman, Jan-Olov

PY - 1996

Y1 - 1996

N2 - 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 105 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 s1. Km passed through a broad minimum in the neutral pH range with values approaching 107 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·Mg 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-(-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 dUTPase·dUDP complex.

AB - 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 105 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 s1. Km passed through a broad minimum in the neutral pH range with values approaching 107 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·Mg 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-(-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 dUTPase·dUDP complex.

M3 - Article

VL - 271

SP - 24010

EP - 24016

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 39

ER -