TY - JOUR
T1 - Heteronuclear NMR and crystallographic studies of wild-type and H187Q Escherichia coli uracil DNA glycosylase
T2 - Electrophilic catalysis of uracil expulsion by a neutral histidine 187
AU - Drohat, Alexander C.
AU - Xiao, Gaoyi
AU - Tordova, Maria
AU - Jagadeesh, Jaya
AU - Pankiewicz, Krzysztof W.
AU - Watanabe, Kyoichi A.
AU - Gilliland, Gary L.
AU - Stivers, James T.
N1 - Copyright:
Copyright 2007 Elsevier B.V., All rights reserved.
PY - 1999/9/14
Y1 - 1999/9/14
N2 - 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) <5.5). These NMR experiments also show that His187 is held in the N(ε2)-H tautomeric form, consistent with the crystallographic observation of a 2.9 Å hydrogen bond from the backbone nitrogen of Ser189 to the ring N(δ1) 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 His187 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 His187.
AB - 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) <5.5). These NMR experiments also show that His187 is held in the N(ε2)-H tautomeric form, consistent with the crystallographic observation of a 2.9 Å hydrogen bond from the backbone nitrogen of Ser189 to the ring N(δ1) 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 His187 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 His187.
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U2 - 10.1021/bi9910880
DO - 10.1021/bi9910880
M3 - Article
C2 - 10508390
AN - SCOPUS:0033554395
SN - 0006-2960
VL - 38
SP - 11876
EP - 11886
JO - Biochemistry
JF - Biochemistry
IS - 37
ER -