Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant.
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 (Km = 5 microM), neutral esters, and at high concentrations of acetylthiocholine, propionylthiocholine, and butyrylthiocholine. However, at low substrate concentrations Km values for acetylthiocholine and succinyldithiocholine were 2-6-fold higher for the fluoride-2 variant. pH rate profiles revealed small differences in pKa 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 degrees C. Affinity of the fluoride-2 variant for sodium fluoride, tacrine, dibucaine, amodiaquin, and succinyldicholine was lower than for usual enzyme. Apparent Ki for succinyldicholine was 125 microM for the fluoride-2 variant and 20 microM for the usual enzyme. Organophosphate inhibition showed equivalent reactivity, indicating that the point mutation altered only the binding properties of the variant. Thus, Km and Ki changes explain the succinyldicholine sensitivity of people carrying the fluoride-2 variant.
Masson
P
P
Centre de Recherches du Service de Santé des Armées, Unité de Biochimie, La Tronche, France.
Adkins
S
S
Gouet
P
P
Lockridge
O
O
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D005459:Fluorides; D014644:Genetic Variation; D005998:Glycine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology; D013379:Substrate Specificity; D013770:Tetraisopropylpyrophosphamide / Q000494:pharmacology; D014162:Transfection; D014633:Valine
D003062:Codon; D011994:Recombinant Proteins; D013770:Tetraisopropylpyrophosphamide; D012969:Sodium Fluoride; D002091:Butyrylcholinesterase; D014633:Valine; D005459:Fluorides; D005998:Glycine