Catalysis by Acetylcholinesterase in Two-Hydronic-Reactive States and Isotope Effects.

2 Low 2 H20 effects 1.0-1.5 for the parameter kcatKm in the hydrolysis of various substrates by acetylcholinesterase is due to normal 2 H20 effects 1.8-2.8 for the parameter kcat and 2 H20 effects of 1.0-2.5 for the parameter Km. The analysis and interpretations of 2 H20 effects in the literature utilizing the parameter kcatKa, which lead to the proposal of isotope insensitivity of the catalytic steps and the hypothesis of a rate-limiting substrate induced-fit conformational change, are incorrect. Since kcat, is the only parameter that can represent the hydron-transfer step solely, the 2 H20 effect can most appropriately be evaluated by using this parameter. The improved binding -0.84 to -2.09 kjmol in H20 obscures the normal 2 H20 effect on kcat when the ratio kcatKm is utilized. Consistent with this, reversible inhibition constant, KIcom, for phenyltrimethylammonium lead to K, 24.5 - 3.5 PM and KI 39 - 3 uM, a 2H20 effect of 1.59 - 0.26. pH-dependence of and kcatKmin 2H20 are subject to variability of the pKapp values, as evaluated in terms of the two-hydronic-reactive states EH and EH2 of AcChE. Such effects are result of uneven decrease in 2H2O of the kinetic parameters kcat, kcatKm for the EH2 state relative to kcat, kcatkm for the EH state, leading to variable shifts in pKapp between 0.5 and 1.2 pH units.