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.

摘要: The nature of the putative general acid His187 in reaction catalyzed by Escherichia coli uracil DNA glycosylase (UDG) was investigated using X-ray crystallography and NMR spectroscopy. crystal structures H187Q UDG, its complex with uracil, have been solved at 1.40 1.60 A resolution, respectively. are essentially identical to those wild-type enzyme, except that side chain Gln187 is turned away from base cannot interact O2. This result provides a structural basis for similar kinetic properties H187A enzymes. ionization state directly addressed 1H−15N experiments optimized histidine ring spin systems, which established neutral catalytically active enzyme (pKa <5.5). These also show held Ne2−H tautomeric form, consistent crystallographic observation 2.9 hydrogen bond backbone nitrogen ...