Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase

Patrick Masson, Pierre Legrand, Cynthia F. Bartels, Marie Thérese Froment, Lawrence M. Schopfer, Oksana Lockridge

Research output: Contribution to journalArticle

120 Scopus citations

Abstract

The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 Å from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in K(m) for compounds with a single positive charge but a 100-fold increase in K(m) for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme- substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in K(m) in the D70G mutant.

Original languageEnglish (US)
Pages (from-to)2266-2277
Number of pages12
JournalBiochemistry
Volume36
Issue number8
DOIs
StatePublished - Feb 25 1997

ASJC Scopus subject areas

  • Biochemistry

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