Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad

Publication date: Available online 19 April 2019Source: Chemico-Biological InteractionsAuthor(s): Bella L. Grigorenko, Dana A. Novichkova, Sofya V. Lushchekina, Irina V. Zueva, Lawrence M. Schopfer, Alexander V. Nemukhin, Sergey D. Varfolomeev, Oksana Lockridge, Patrick MassonAbstractA computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198·H438·E325) was replaced by S198·H438·N322E in silico. Molecular dynamics for 1.5 μs and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293 cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293 cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after p...
Source: Chemico Biological Interactions - Category: Biochemistry Source Type: research