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

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 re solution, respectively. The structures are essentially identical to those o f 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 provi des a structural basis for the similar kinetic properties of the H187Q and H187A enzymes. The ionization state of His187 was directly addressed with H -1-N-15 NMR experiments optimized for histidine ring spin systems, which es tablished that His 187 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 N-epsilon 2-H tautomeric form, consistent with the crystallographic ob servation of a 2.9 Angstrom hydrogen bond from the backbone nitrogen of Ser 189 to the ring N-delta 1 of His187. The energetic cost of breaking this hy drogen bond may contribute significantly to the low pK(a) of His 187. 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 b ase catalysis by Asp64 and electrophilic stabilization of the developing en olate on uracil O2 by a neutral His187.