NITROGEN ISOTOPE EFFECTS ON ACETYLCHOLINESTERASE-CATALYZED HYDROLYSISOF OMICRON-NITROACETANILIDE

The nitrogen-15 isotope effect on V/K for Electrophorus electricus ace tylcholinesterase-catalyzed hydrolysis of o-nitroacetanilide has been determined by isotope ratio mass spectrometry. The effect determined i n buffered H2O (0.1 M sodium phosphate, 0.1 N NaCl, pH 7.3, 25-degrees -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 equivalentl y buffered D2O (pD = 7.7), 15V/K = 1.0106 +/- 0.0002. The correspondin g solvent isotope effect is (D)V/K = 1.56 +/- 0.03. The solvent isotop e effect on the nitrogen isotope effect is interpreted in terms of a m echanism 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 re latively narrow limits to be placed on the isotope effects for the che mical steps. The solvent and substrate isotope effects for the formati on of the tetrahedral intermediate are (D)k5 = 2.6-3.7 and 15k5 = 1.00 0-1.009, respectively. The corresponding isotope effects for the decom position of the intermediate are (D)k7 = 1.0-1.5 and 15k7' = 15k5(15)k 7/15k6 = 1.027-1.053. The value of 15k7' is consistent with a transiti on state for decomposition of the tetrahedral intermediate in which C- N bond breaking is occurring.