engleski

Inhibition of acetylcholinesterase and butyrylcholinesterase mutants by the pyridinium oximes 2-PAM and HI-6

2-PAM (2-(hydroxyiminomethyl)-1-methylpyridinium chloride) and HI-6 ((1-(2’ -hydroxyiminomethyl-1'-pyridinium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane dichloride)) are reversible inhibitors of acetylcholinesterase (AChE ; EC 3.1.1.7) and butyrylcholinesterase (BChE ; EC 3.1.1.8). 2-PAM and HI-6, as strong nucleophiles, are also efficient reactivators of phosphylated AChE and BChE. In attempting to determine the amino acid residues within the active site gorge involved in the interaction with the oximes, selective mutants of mouse AChE were subjected to inhibition by 2-PAM and HI-6 and their affinities were compared with wild-type AChE and BChE. Mutations in the choline binding site (Y337A) or combined with acyl pocket mutations (F295L/Y337A, F297I/Y337A) were employed to enlarge active site gorge dimensions and to mimic BChE active site. Enzyme-oxime dissociation constants (Ki) for the catalytic site were evaluated from the apparent dissociation constants as a function of the substrate concentration (0.05-1.0 mM acetylthiocholine). Dissociation constants for AChE w.t. were 150 and 47 µ ; ; ; ; M, for BChE w.t. 320 and 23 µ ; ; ; ; M for 2-PAM and HI-6, respectively. Introduced mutations lowered oxime binding affinities for both oximes, and Ki were 590 and 87 µ ; ; ; ; M for Y337A, 650 and 110 µ ; ; ; ; M for F295L/Y337A, and 1700 and 180 µ ; ; ; ; M for F297I/Y337A, for 2-PAM and HI-6, respectively. Despite introduced mutations in AChE, which correspond to residues found in BChE active site, affinities for the oximes did not approximate BChE affinities. This might imply that binding of HI-6 and 2-PAM did not include their stabilization with residues 337, 295 and 297. However, from the calculated change of free energy of binding, it seems that mutations Y337A and F297I affected binding with a cumulative effect, since the calculated change of energy was nearly doubled for F297I/Y337A relative to the single mutation Y337A, or to F295L/Y337A. (Supported by grant No: 22014, Ministry of Science, Education and Sports, Croatia)

inhibition; acetylcholinesterase; butyrylcholinesterase; mutants; antidots; oxime; 2-PAM; HI-6

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