Nitrogen isotope effects on acetylcholinesterase-catalyzed hydrolysis of o-nitroacetanilide

Article abstract of DOI:10.1021/ja00078a003

The nitrogen-15 isotope effect on V/K for Electrophorus electricus acetylcholinesterase-catalyzed hydrolysis of o-nitroacetanilide has been determined by isotope ratio mass spectrometry. The effect determined in buffered H₂O (0.1 M sodium phosphate, 0.1 N NaCl, pH 7.3, 25 °C) is ¹⁵V/K = 1.0119 ± 0.0005. A small though palpable decrease of the isotope effect is observed when the reaction is run in equivalently buffered D₂O (pD = 7.7), ¹⁵V/K = 1.0106 ± 0.0002. The corresponding solvent isotope effect is ^DV/K = 1.56 ± 0.03. The solvent isotope effect on the nitrogen isotope effect is interpreted in terms of a mechanism in which successive transition states for induced fit and for formation and decomposition of a uninegative tetrahedral intermediate contribute to rate determination of V/K. Numerical modeling allows relatively narrow limits to be placed on the isotope effects for the chemical steps. The solvent and substrate isotope effects for the formation of the tetrahedral intermediate are ^DK₅ = 2.6-3.7 and ¹⁵k₅ = 1.000-1.009, respectively. The corresponding isotope effects for the decomposition of the intermediate are ^Dk₇ = 1.0-1.5 and ¹⁵k₇′ = ¹⁵k₅¹⁵k₇/¹⁵k₆ = 1.027-1.053. The value of ¹⁵k₇′ is consistent with a transition state for decomposition of the tetrahedral intermediate in which C-N bond breaking is occurring.