Amino acids residues involved in stereoselective inhibition of cholinesterase with bambuterol (CROSBI ID 528461)
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Podaci o odgovornosti
Bosak, Anita ; Kovarik, Zrinka ; Gazić, Ivana ; Vinković, Vladimir
engleski
Amino acids residues involved in stereoselective inhibition of cholinesterase with bambuterol
Bambuterol is a chiral carbamate that inhibits butyrylcholinesterase (BChE) about 16, 000 times faster than acetylcholinesterase (AChE) (1). Regardless of bambuterol's own selectivity, both enzymes have a stereoselective preference for R-bambuterol (2). In order to relate stereoselectivity of cholinesterases and bambuterol selectivity to the active site residues, we studied the time course of inhibition of recombinant mouse enzymes BChE w.t., AChE w.t. and AChE mutants by R- and S-bambuterol. Mutations in the choline binding site (Y337A) combined with that in the acyl pocket (F295L/Y337A, F297I/Y337A, F295L/F297I/Y337A) or mutations in the peripheral site (Y124Q) were employed to mimic BChE active site residues. All studied cholinesterases were progressively inhibited by both bambuterol enantiomers, displaying preference for the R- over S-enantiomer: about four times for the wild type enzymes, 12-16 times for Y337A and the double mutants, and only 1.7 times for F295L/F297I/Y337A and Y124Q. Stereoselectivity was increased through the enlargement of the AChE choline binding site and of the acyl pocket by single or double mutations, which resulted in increased inhibition rate constants of up to 3000 and 950 times over the rate of the wild-type AChE with R-bambuterol and S-bambuterol, respectively. Low stereoselectivity of the peripheral single-site mutant, Y124Q, is due to increased inhibition rate by S-bambuterol. It seems that this residue change attenuated the difference in configuration between bambuterol enantiomers. When three aromatic residues were mutated (F295L/F297I/Y337A), both enantiomers became poor inhibitors, because inhibition rates rapidly decreased in comparison to that of the mutants and AChE w.t. Although four out of five AChE mutants were inhibited at rates that were closer to BChE than to AChE, selectivity cannot be predicted simply on the basis of the enlarged volume of the gorge made by mutating AChE to mimic BChE active site residues. 1. Kovarik et al. 1999 Biochim. Biophys. Acta 1422:261-271. 2. Gazić et al. 2006 Anal. Bioanal. Chem. 385:1 513-1519.
cholinesterase; bambuterol; enantiomers; inhibition; stereoselectivity; mutants
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Podaci o prilogu
114-x.
2007.
objavljeno
Podaci o matičnoj publikaciji
The IXth International Meeting on Cholinesterases, Suzhou, Kina, Program book
Podaci o skupu
The IXth International Meeting on Cholinesterases
poster
06.05.2007-10.05.2007
Suzhou, Kina