Kinetic Isotope Effect Studies of the Reaction Catalyzed by Uracil DNA Glycosylase: Evidence for an Oxocarbenium Ion-Uracil Anion Intermediate
R M. Werner, J T. Stivers
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.
kenetic isotope effects, transition state features, uracil DNA glycosylase
and Stivers, J.
Kinetic Isotope Effect Studies of the Reaction Catalyzed by Uracil DNA Glycosylase: Evidence for an Oxocarbenium Ion-Uracil Anion Intermediate, Biochemistry
(Accessed September 22, 2023)