Simple General Acid-Base Catalysis and Virtual Transition States for Acetylcholinesterase-Catalyzed Hydrolysis of Phenyl Esters

Article abstract of DOI:10.1021/ja00235a036

Acetylcholinesterase-catalyzed hydrolyses of the acetyl esters phenyl acetate and o-nitrophenyl acetate and of the chloroacetyl esters phenyl chloroacetate and p-methoxyphenyl chloroacetate have been investigated .V's are quantitatively similar for the constituents of each pair of esters, which indicates that deacylation is partly rate limiting when the enzyme is saturated by substrate.Solvent deuterium isotope effects for V/K are near unity, which is consistent with virtual acylation transition states that are prominently rate limited by nonchemical events .On the other hand, solvent deuterium isotope effects for V fall in the range 1.6-2.26 and are interpreted in terms of transition states that are stabilized by solvation catalytic proton bridges.The Eyring plot constructed from initial velocities of AChE-catalyzed hydrolysis of o-nitrophenyl acetate at ₀ >> K is nonlinear downward and is interpreted in terms of prominent rate determination from both acylation and deacylation.However, the solvent isotope effect for the reaction is independent of temperature, which indicates that the solvent isotope effects for the acylation and deacylation components of V must be of comparable magnitude.Proton inventory plots of partial solvent isotope effects on initial velocities at ₀ >> K vs. the atom fraction of deuterium in mixed H2O-D2O buffers are linear for the substrates phenyl chloroacetate and o-nitrophenyl acetate.Therefore, AChE behaves as a simple general acid-base catalyst for the studied ester hydrolyses. pL-rate profiles (L = H, D) for hydrolysis of o-nitrophenyl acetate are sigmoidal in shape, and nonlinear-least-squares analysis gives pK_a^H2O = 6.31 +/- 0.03, pK_a^D2O = 6.81 +/- 0.03, and ^D2OV_i,lim = 1.82 +/- 0.02.The β-deuterium secondary isotope effect for o-nitrophenyl acetate hydrolysis is ^D3V_i = 0.96₀ +/- 0.01₇.These results are interpreted in terms of a virtual transition for AChE-catalyzed ester hydrolysis that is a weighted average of acylation and deacylation transition states that are each stabilized by single-proton transfers.