Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant

P. Masson, S. Adkins, P. Gouet, O. Lockridge

Research output: Contribution to journalArticle

42 Scopus citations

Abstract

Kinetics of recombinant fluoride-2 variant of human butyrylcholinesterase (Gly390 Val) secreted by Chinese hamster ovary cells were compared to recombinant usual and to usual butyrylcholinesterase purified from human plasma. The usual and fluoride-2 variant were indistinguishable with regard to hydrolysis of benzoylcholine (K(m) = 5 μM), neutral esters, and at high concentrations of acetylthiocholine, propionylthiocholine, and butyrylthiocholine. However, at low substrate concentrations K(m) values for acetylthiocholine and succinyldithiocholine were 2-6-fold higher for the fluoride-2 variant. pH rate profiles revealed small differences in pK(α) that could be attributed to changes in the active site histidine environment. On the other hand, Arrhenius plot analysis of o-nitrophenylbutyrate hydrolysis at pH 7.5 showed no difference in activation energy between fluoride-2 and usual butyrylcholinesterases. Both exhibited an anomalous temperature dependence with a wavelike change in activation energy around 18 °C. Affinity of the fluoride-2 variant for sodium fluoride, tacrine, dibucaine, amodiaquin, and succinyldicholine was lower than for usual enzyme. Apparent K(i) for succinyldicholine was 125 μM for the fluoride-2 variant and 20 μM for the usual enzyme. Organophosphate inhibition showed equivalent reactivity, indicating that the point mutation altered only the binding properties of the variant. Thus, K(m) and K(i) changes explain the succinyldicholine sensitivity of people carrying the fluoride-2 variant.

Original languageEnglish (US)
Pages (from-to)14329-14341
Number of pages13
JournalJournal of Biological Chemistry
Volume268
Issue number19
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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