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

Autor: Grigorenko BL; N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia; Lomonosov State University, Chemistry Department, Moscow 119991, Russia., Novichkova DA; N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia., Lushchekina SV; N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia. Electronic address: sofya.lushchekina@gmail.com., Zueva IV; Arbuzov Institute of Organic and Physical Chemistry, FRC Kazan Scientific Center of RAS, Arbuzov Str. 8, Kazan, 420088, Russia; Kazan Federal University, Neuropharmacology Laboratory, Kremlevskaya Str, 18, Kazan, 420008, Russia., Schopfer LM; Eppley Institute, University of Nebraska Medical Center, Omaha, NE, 68198, USA., Nemukhin AV; N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia; Lomonosov State University, Chemistry Department, Moscow 119991, Russia., Varfolomeev SD; N.M. Emanuel Institute of Biochemical Physics of Russian Academy of Sciences, Kosygina Str. 4, Moscow 119334, Russia; Lomonosov State University, Chemistry Department, Moscow 119991, Russia., Lockridge O; Eppley Institute, University of Nebraska Medical Center, Omaha, NE, 68198, USA., Masson P; Kazan Federal University, Neuropharmacology Laboratory, Kremlevskaya Str, 18, Kazan, 420008, Russia. Electronic address: pym.masson@free.fr.
Jazyk: angličtina
Zdroj: Chemico-biological interactions [Chem Biol Interact] 2019 Jun 01; Vol. 306, pp. 138-146. Date of Electronic Publication: 2019 Apr 19.
DOI: 10.1016/j.cbi.2019.04.019
Abstrakt: 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.
(Copyright © 2019 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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