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
Ac. Drohat et al., 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, BIOCHEM, 38(37), 1999, pp. 11876-11886
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.