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J Biol Chem, Vol. 273, Issue 31, 19509-19517, July 31, 1998
From the Departments of The contribution of the oxyanion
hole to the functional architecture and to the hydrolytic efficiency of
human acetylcholinesterase (HuAChE) was investigated through single
replacements of its elements, residues Gly-121, Gly-122 and the
adjacent residue Gly-120, by alanine. All three substitutions resulted
in about 100-fold decrease of the bimolecular rate constants for
hydrolysis of acetylthiocholine; however, whereas replacements of
Gly-120 and Gly-121 affected only the turnover number, mutation of
residue Gly-122 had an effect also on the Michaelis constant. The
differential behavior of the G121A and G122A enzymes was manifested
also toward the transition state analog
m-(N,N,N-trimethylammonio)trifluoroacetophenone
(TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward
TMTFA was 2000-11,000-fold lower than that of the wild type HuAChE;
however, the G121A enzyme exhibited a rapid inhibition pattern, as
opposed to the slow binding kinetics shown by the G122A enzyme. For
both phosphates (diethyl phosphorofluoridate, diisopropyl
phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman),
the decrease in inhibitory activity toward the G121A enzyme was very
substantial (2000-6700-fold), irrespective of size of the alkoxy
substituents on the phosphorus atom. On the other hand, for the G122A
HuAChE the relative decline in reactivity toward phosphonates
(500-460-fold) differed from that toward the phosphates (12-95-fold).
Although formation of Michaelis complexes with substrates does not seem
to involve significant interaction with the oxyanion hole, interactions
with this motif are a major stabilizing element in accommodation of
covalent inhibitors like organophosphates or carbamates. These
observations and molecular modeling suggest that replacements of
residues Gly-120 or Gly-121 by alanine alter the structure of the
oxyanion hole motif, abolishing the H-bonding capacity of residue at
position 121. These mutations weaken the interaction between HuAChE and
the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in
reactivity due to replacement of residue Gly-122 seem to result from
steric hindrance at the active center acyl pocket.
Functional Characteristics of the Oxyanion Hole in Human
Acetylcholinesterase
,,,, and
Biochemistry & Molecular
Biology and § Organic Chemistry, Israel Institute for
Biological Research, Ness-Ziona, 70450, Israel
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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