engleski

Reversible inhibition of acetylcholinesterase and butyrylcholinesterase by a coumarin derivative and by 4,4'-bipyridine

It was shown earlier that the coumarin derivative 3-chloro-7-hydroxy-4-methylcoumarin (CHMC) is an allosteric inhibitor of acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; 3.1.1.8) while 4,4'-bipyridine (4,4'-BP) binds to both the catalytic and allosteric site of the enzyme. Binding of the CHMC and 4,4'-BP to recombinant mouse w.t. AChE and BChE, to peripheral site-directed AChE mutants and to human serum BChE variants has now been studied. The enzyme activity was measured with acetylthiocholine as substrate. Enzyme/inhibitor dissociation constants for the catalytic and peripheral sites were evaluated from the apparent dissociation constants as a function of the substrate concentration (0.1 -10 mM). The competition between substrate and CHMC displayed two binding sites on AChE mutants Y72N, Y124Q, W286A and W286R, and the atypical and fluoride-resistant BChE variants. The dissociation constants for the peripheral site (from 0.06 to 0.1 mM) were on average two times higher than for the catalytic site (0.02 - 0.06 mM). CHMC displayed binding only to the catalytic site (Ka = 0.1 mM) of Y72N/Y124Q/W286A mutant and only to the peripheral site of AChE w.t. and usual BChE . 4,4'-BP bound to both sites of the AChE mutant Y72N/Y124Q/W286A, AChE w.t. and BChE w.t.. The two compounds had the lowest affinity for the Y72N/Y124Q/W286A mutant and displayed the most pronounced competition with acetylthiocholine over the studied substrate concentration range. However, the Michaelis constant of the mutant was similar to those of the other enzymes. The enhancement of competition might therefore be attributed, in part, to the differential effect of mutations on the binding of the substrate and the compounds CHMC or 4,4'-BP.

acetylcholinesterase; butyrylcholinesterase; 4;4'-bipyridine; coumarin derivative; reversible inhibition; molecular modelling

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