Nitrogen Isotope Effects on Acetylcholinesterase-Catalyzed Hydrolysis of o-Nitroacetanilide

Muralikrishna Rao, Paul N. Barlow, Alton N. Pryor, Daniel M. Quinn, Pyotr Paneth, Marion H. O’Leary, W. Phillip Huskey

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Abstract

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 [formula omitted] . The value of [formula omitted] is consistent with a transition state for decomposition of the tetrahedral intermediate in which C-N bond breaking is occurring.

Original languageEnglish (US)
Pages (from-to)11676-11681
Number of pages6
JournalJournal of the American Chemical Society
Volume115
Issue number25
DOIs
StatePublished - Dec 1 1993

All Science Journal Classification (ASJC) codes

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry

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