FUNCTIONAL-CHARACTERISTICS OF THE OXYANION HOLE IN HUMAN ACETYLCHOLINESTERASE

The contribution of the oxyanion hole to the functional architecture a nd to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residue s Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine, All t hree substitutions resulted in about 100-fold decrease of the bimolecu lar rate constants for hydrolysis of acetylthiocholine; however, where as replacements of Gly-120 and Gly-121 affected only the turnover numb er, mutation of residue Gly-122 had an effect also on the Michaelis co nstant. The differential behavior of the G121A and G122A enzymes was m anifested also toward the transition state analog m-(N,N,N-trimethylam monio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, car bamates, and toward selected noncovalent active center ligands, Reacti vity 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 t he G122A enzyme. For both phosphates (diethyl phosphorofluoridate, dii sopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin an d soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000-6700-fold), irrespective of size of the alk oxy substituents on the phosphorus atom, On the other hand, for the G1 22A HuAChE the relative decline in reactivity toward phosphonates (500 -460-fold) differed from that toward the phosphates (12-95-fold). Alth ough 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 co valent inhibitors like organophosphates or carbamates. These observati ons and molecular modeling suggest that replacements of residues Gly-1 20 or Gly-121 by alanine alter the structure of the oxyanion hole moti f, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuA-ChE and the various liga nds 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.