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

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 primary 13C KIE (=1.010 ± 0.009) and the large secondary α-deuterium KIE (=1.201 ± 0.021) indicate that (i) the glycosidic bond is essentially completely broken in the transition state and (ii) there is significant sp2 character at the anomeric carbon. Large secondary β-deuterium KIEs were observed when [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 β-deuterium KIEs indicate strong hyperconjugation between the elongated glycosidic bond and both of the C2‘−H2‘ bonds. Geometric interpretation of these β-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.