Functional characteristics of the oxyanion hole in human acetylcholinesterase.
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.
Department of Biochemistry & Molecular Biology, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel.
A
A
Ordentlich
D
D
Barak
C
C
Kronman
N
N
Ariel
Y
Y
Segall
B
B
Velan
A
A
Shafferman
D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D002219:Carbamates / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011729:Pyridostigmine Bromide / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity
D002219:Carbamates; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D009943:Organophosphorus Compounds; D010710:Phosphates; D011994:Recombinant Proteins; D000110:Acetylcholinesterase; D011729:Pyridostigmine Bromide