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 in: Biochemistry (Easton) 1999-09, Vol.38 (37), p.11876-11886
• Main Authors: Drohat, Alexander C, Xiao, Gaoyi, Tordova, Maria, Jagadeesh, Jaya, Pankiewicz, Krzysztof W, Watanabe, Kyoichi A, Gilliland, Gary L, Stivers, James T
• Format: Article
• Language: English
• Subjects: Binding Sites - genetics; Carbon Isotopes; Catalysis; Crystallography, X-Ray; DNA Glycosylases; Enzyme Stability; Escherichia coli; Escherichia coli - enzymology; Glutamine - genetics; Histidine - chemistry; Histidine - genetics; Histidine - metabolism; Hydrogen Bonding; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; N-Glycosyl Hydrolases - chemistry; N-Glycosyl Hydrolases - genetics; N-Glycosyl Hydrolases - metabolism; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Protons; Substrate Specificity; Uracil - chemistry; Uracil - metabolism; Uracil-DNA Glycosidase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi9910880

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 Å resolution, respectively. The structures are essentially identical to those of the wild-type enzyme, except that the side chain of Gln187 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