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

September 14, 1999

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 (pK_a <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 pK_a 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

Pub Type

Journals

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 December 26, 2024)