Kinetic Isotope Effect Studies of the Reaction Catalyzed by Uracil DNA Glycosylase: Evidence for an Oxocarbenium Ion-Uracil Anion Intermediate

Published

November 1, 2000

Author(s)

R M. Werner, J T. Stivers

Abstract

The DNA repair enzyme uracil DNA glycosylase catalyzes the first step in the uracil base excision repair pathway, the hydrolytic cleavage of the N-glycosidic bond of deoxyuridine in DNA. Here we report kinetic isotope effect (KIE) measurements that have allowed the determination of the transition-state structure for this important reaction. The small 1o 13C KIE = 1.010 0.009, and the large a-2o 2H KIE = 1.201 0.021 indicate that (i) the glycosidic bond is essentially completely broken in the transition state and (ii) that there is a significant sp2 character at the anomeric carbon. Large secondary b-deuterium KIEs were observed with [2 R -2H] = 1.102 0.011, and [2 S -2H] = 1.106 0.010. The nearly equal and large magnitudes of the two stereospecific b-deuterium KIEs indicates strong hyperconjugation between the elongated glycosidic bond and both of the C2 -H2 bonds. Geometric interpretation of these b-deuterium KIEs indicates that the furanose ring adopts a mild 3' exo sugar pucker in the transition-state, as would be expected for maximal stabilization of an oxocarbenium ion. Taken together, these results strongly indicate that the reaction proceeds through a dissociative transition state, with complete dissociation of the uracil anion followed by addition of water. To our knowledge this is the first transition-state structure determined for enzymatic cleavage of the glycosidic linkage in a pyrimidine deoxyribonucleotide.

Citation

Biochemistry

Volume

39

Issue

No. 46

Pub Type

Journals

Keywords

kenetic isotope effects, transition state features, uracil DNA glycosylase

Citation

Werner, R. and Stivers, J. (2000), Kinetic Isotope Effect Studies of the Reaction Catalyzed by Uracil DNA Glycosylase: Evidence for an Oxocarbenium Ion-Uracil Anion Intermediate, Biochemistry (Accessed December 12, 2024)