Uracil DNA glycosylase (UDG) cleaves the glycosidic bond of deoxyuridine in DNA using a hydrolytic mechanism, with an overall catalytic rate enhancement of 1012-fold over the solution reaction. The nature of the enzyme-substrate interactions that lead to this large rate enhancement are key to understanding enzymatic DNA repair. Using 1H and heteronuclear NMR spectroscopy, we have characterized one such interaction in the ternary product complex of Escherichia coli UDG, the short (2.7 Å) H bond between His187 N(ε)2 and uracil O2. The H bond proton is highly deshielded at 15.6 ppm, indicating a short N-O distance and exhibits a solvent exchange rate that is 400- and 105-fold slower than free imidazole at pH 7.5 and pH 10, respectively. Heteronuclear NMR experiments at neutral pH show that this H bond involves the neutral imidazole form of His187 and the N1-O2 imidate form of uracil. The excellent correspondence of the pK(a) for the disappearance of the H bond (pK(a) = 6.3 ± 0.1) with the previously determined pK(a) = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil 02 [Drohat, A. C., and Stivers, J. T. (2000) J. Am. Chem. Soc. 122, 1840-1841]. Although the above characteristics suggest a short strong H bond, the D/H fractionation factor of φ = 1.0 is more typical of a normal H bond. This unexpected observation may reflect a large donor-acceptor pK(a) mismatch or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (φ < 1) and increased bending frequencies (φ > 1) relative to the O-H bonds of water. The role of this H bond in catalysis by UDG and several approaches to quantify the H bond energy are discussed.
ASJC Scopus subject areas