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 butyrylcholinester ase (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 muM), neutral e sters, and at high concentrations of acetylthiocholine, propionylthioc holine, and butyrylthiocholine. However, at low substrate concentratio ns K(m) values for acetylthiocholine and succinyldithiocholine were 2- 6-fold higher for the fluoride-2 variant. pH rate profiles revealed sm all differences in pK(a) that could be attributed to changes in the ac tive site histidine environment. On the other hand, Arrhenius plot ana lysis of o-nitrophenylbutyrate hydrolysis at pH 7.5 showed no differen ce in activation energy between fluoride-2 and usual butyrylcholineste rases. Both exhibited an anomalous temperature dependence with a wavel ike change in activation energy around 18-degrees-C. Affinity of the f luoride-2 variant for sodium fluoride, tacrine, dibucaine, amodiaquin, and succinyldioholine was lower than for usual enzyme. Apparent K(i) for succinyldicholine was 125 muM for the fluoride-2 variant and 20 mu M for the usual enzyme. Organophosphate inhibition showed equivalent r eactivity, indicating that the point mutation altered only the binding properties of the variant. Thus, K(m) and K(i) changes explain the su ccinyldicholine sensitivity of people carrying the fluoride-2 variant.