engleski

Amino acid residues involved in cholinesterase inhibition with metaproterenol and its bisdimethylcarbamate

Introduction: Selectivity of cholinesterases and their interaction with various compounds, especially drugs, is a subject of many studies in the field of biochemistry and pharmacology, due to important roles of cholinesterases in organism and their involvement in metabolism of many drugs. Metaproterenol is a bronchodilator used in the treatment of asthma. Metacarb (bisdimethylcarbamate of metaproterenol) is structurally related to bambuterol. Bambuterol, a prodrug of bronchodilator terbutaline and one of the most selective butyrylcholinesterase (BChE) inhibitor. Metacarb, like bambuterol, carbamylates the active site serine and progressively inhibits cholinesterases. Metaproterenol, a final product of decarbamylation, is reversible cholinesterase inhibitor. The aim of the work was to identify amino acids that govern the inhibition with metaproterenol and metacarb by determining dissociation (KI) and inhibition rate (ki) constants, respectively. Results and discussion: Metaproterenol and metacarb were studied as inhibitors of mouse recombinant acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and six AChE mutants. Mutations in the choline-binding site (Y337A) combined with those in the acyl pocket (F295L/Y337A, F297I/Y337A, and F295L/F297I/Y337A) or those in the peripheral site (Y124Q and Y72N/Y124Q/W286R) were employed to mimic BChE active site residues. Competitive inhibition was observed for AChE w.t., BChE w.t., choline binding site and acyl pocket mutants, while non-competitive inhibition was noticed in case of peripheral site mutants. All studied cholinesterases displayed poor affinity (1/KI) for metaproterenol binding (KI=0.32 – 13 mM). However, even than 3 times higher affinity of BChE than AChE was observed. Although mutations in choline binding site and acyl pocket mimics BChE, affinities of these mutants were up to 4 times higher than that of BChE. Peripheral site mutants had the same or a 3 times lower affinity than AChE w.t. The inhibition rate constant of BChE inhibition by metacarb was 280 times higher than that of AChE. All mutations employed, except those including F297I, increased inhibition rates comparing to AChE w.t. (up to 30-times for F295L/Y337A and Y124Q) and therefore decreased selectivity seen for the wild type enzymes. Conclusions: Cholinesterase selectivity for metaproterenol reversible binding and carbamylation selectivity of metacarb is dictated with amino residues in choline-binding site and peripheral site residues.

metaproterenol; carbamate; metacarb; selectivity; cholinesterase; mutants; inhibition

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