TY - JOUR
T1 - Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad
AU - Grigorenko, Bella L.
AU - Novichkova, Dana A.
AU - Lushchekina, Sofya V.
AU - Zueva, Irina V.
AU - Schopfer, Lawrence M.
AU - Nemukhin, Alexander V.
AU - Varfolomeev, Sergey D.
AU - Lockridge, Oksana
AU - Masson, Patrick
N1 - Publisher Copyright:
© 2019 Elsevier B.V.
PY - 2019/6/1
Y1 - 2019/6/1
N2 - A 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 phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.
AB - A 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 phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198·H438·N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.
KW - Butyrylcholinesterase
KW - Catalytic triad
KW - Computer-designed enzyme
KW - Markov state model
KW - Molecular dynamics
KW - Organophosphate inhibition/reactivation
KW - QM/MM
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U2 - 10.1016/j.cbi.2019.04.019
DO - 10.1016/j.cbi.2019.04.019
M3 - Article
C2 - 31009643
AN - SCOPUS:85064686886
SN - 0009-2797
VL - 306
SP - 138
EP - 146
JO - Chemico-Biological Interactions
JF - Chemico-Biological Interactions
ER -