Escherichia Coli Uracil DNA Glycosylase: NMR Characterization of the Short Hydrogen Bond From His 187 to Uracil 02

Published

October 1, 2000

Author(s)

A C. Drohat, J T. Stivers

Abstract

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 ) H bond between His187 N^e2 and uracil O2. The H bond proton is highly desheilded 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 pKa for disappearance of the H bond (pKa = 6.3 0.1) with the previously determined pKa = 6.4 for the N1 proton of enzyme-bound uracil indicates that the H bond requires negative charge on uracil O2 [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 diameter} = 1.0 is more typical of a normal H bond. This unexpected observation may reflect a large donor-acceptor pKa mismatch, or the net result of two opposing effects on vibrational frequencies: decreased N-H bond stretching frequencies (diameter} < 1), and increased bending frequencies (diameter} > 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.

Citation

Biochemistry

Volume

39

Issue

No. 39

Pub Type

Journals

Keywords

H/D fractionation, histidine 187, hydrogen bond energy, NMR spectroscopy, pH dependence, proton exchange rates, short hydrogen bond

Citation

Drohat, A. and Stivers, J. (2000), Escherichia Coli Uracil DNA Glycosylase: NMR Characterization of the Short Hydrogen Bond From His 187 to Uracil 02, Biochemistry (Accessed July 26, 2024)