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Heteronuclear NMR and Crystallographic Studies of Wild-Type and H187Q Escherichia Coli Uracil DNA Glycosylase: Electrophilic Catalysis of Uracil Expulsion by a Neutral Histidine 187

Published

Author(s)

A C. Drohat, G Xiao, M Tordova, J. Jagadeesh, K Pankiewicz, K A. Watanbe, G L. Gilliland, J T. Stivers

Abstract

The nature of the putative general acid, His187, in the reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. The crystal structures of H187Q UDG, and its complex with uracil, have been solved at 1.40 and 1.60 Angstrom}resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln 287 is turned away from the uracil base and cannot interact with uracil O2. This result provides a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with 1H-15N NMR experiments optimized for histidine ring spin systems, which established that His187 is neutral in the catalytically active state of the enzyme (pKa <5.5). These NMR experiments also show that His187 is held in the Nε2-H tautomeric form, consistent with the crystallographic observation of a 2.9 Angstrom} hydrogen bond from the backbone nitrogen of Ser189 to the ring Nδ1 of His187. The energetic cost of breaking this hydrogen bond may contribute significantly to the low pKa of His187. Thus, the traditional view that a cationic His 187 donates a proton to uracil O2 is incorrect. Rather, we propose a concerted mechanism involving general base catalysis by Asp64 and electrophilic stabilization of the developing enolate on uracil O2 by a neutral His 187.
Citation
Biochemistry
Volume
38
Issue
37

Keywords

2'-fluoro-2'deoxyuridine nucleotide, 2-aminopurine, E. coli uracil DNA glycosylase, electrophilic catalysis, neutral histidine

Citation

Drohat, A. , Xiao, G. , Tordova, M. , Jagadeesh, J. , Pankiewicz, K. , Watanbe, K. , Gilliland, G. and Stivers, J. (1999), Heteronuclear NMR and Crystallographic Studies of Wild-Type and H187Q Escherichia Coli Uracil DNA Glycosylase: Electrophilic Catalysis of Uracil Expulsion by a Neutral Histidine 187, Biochemistry (Accessed July 15, 2024)

Issues

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Created September 13, 1999, Updated October 12, 2021