engleski

Inhibition of acetylcholinesterase mutants and butyrylcholinesterase with enantiomers of ethopropazine

Ethopropazine is a phenothiazine class compound and possesses an asymmetrical carbon atom in its structure. After enantioselective separation of enantiomers (E #1 and E #2), kinetic study of ethopropazine cholinesterase inhibition was conducted on mouse wild-type (w.t.) butyrylcholinesterase (BChE ; EC 3.1.1.8), acetylcholinesterase (AChE ; EC 3.1.1.7), and its mutants. Dissociation constants of the enzyme-inhibitor complexes were calculated from the effect of acetylthiocholine concentration (0.1 - 5.0 mM) on the degree of inhibition using the Hunter-Downs plot. Ethopropazine is a potent inhibitor for BChE w.t. and for the mutants that mimic BChE (Y124Q, W286A, Y72N/Y124Q/W286A, and Y72N/Y124Q/W286R). Dissociation constants (Ki) of enzyme-inhibitor complexes for BChE w.t. were 0.08 (M and 0.22 (M for E #2 and E #1, respectively. Dissociation constants for E #1 were three times lower than for E #2 for all of mutants, except the W286A mutant. The inhibition of W286A was the same with both enantiomers (Ki = 2.1 (M). The mutations enlarged the active site gorge in comparison with that of AChE w.t., and new interactions are also formed. Ethopropazine has higher affinity for the mutants which mimic BChE than for AChE w.t. (Ki ( 100 (M). These mutants revealed a non-competitive inhibition at the peripheral binding site, while BChE w.t. displayed a competitive inhibition at the active site. Interaction between aromatic tryptophane 286 and positively charged nitrogen in ethopropazine affects enantiomer differentiation. Stereospecific orientation of enantiomers was eliminated by replacing tryptophane 286 with alanine, an aliphatic amino acid, which is probably the main reason why both enantiomers have the same inhibition constants. The enantiomers were separated at CATBIO Laboratory, Ruđer Bošković Institute, Zagreb-Croatia, and the mutants were a gift from the Department of Pharmacology, University of California, San Diego-USA.

Ethopropazine; butyrylcholinesterase; acetylcholinesterase; reversible inhibition

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