Escherichia coli uracil DNA glycosylase: NMR characterization of the short hydrogen bond from His187 to uracil O2

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.