The role of Phe329 in binding of cationic triarylmethane dyes to human butyrylcholinesterase

• Published in: Archives of biochemistry and biophysics Vol. 511; no. 1; pp. 64 - 68
• Main Authors: Biberoglu, Kevser, Tacal, Özden, Akbulut, Hakan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.07.2011
• Subjects: Amino Acid Substitution; Binding Sites - genetics; biomedical research; Butyrylcholinesterase - chemistry; Butyrylcholinesterase - genetics; Butyrylcholinesterase - metabolism; Cholinesterase inhibition; Cholinesterase Inhibitors - chemistry; Cholinesterase Inhibitors - metabolism; Coloring Agents - chemistry; Coloring Agents - metabolism; enzymes; HEK293 Cells; Human BChE; Humans; industry; Kinetics; Malachite green; Methyl green; Methyl Green - chemistry; Methyl Green - metabolism; Mutagenesis, Site-Directed; mutants; phenylalanine; Phenylalanine - chemistry; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; regression analysis; Rosaniline Dyes - chemistry; Rosaniline Dyes - metabolism; Site-directed mutagenesis; Methyl green; Human BChE; Cholinesterase inhibition; Site-directed mutagenesis; Malachite green
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2011.04.007

► We test the role of five amino acids in the inhibition of human BChE by MG and MeG. ► Phe329 is located on E-helix in human BChE. ► Substitution of F329 by A reveals differences in inhibitory pattern and K i values. ► Phe329 is a critical residue in MG and MeG binding to human BChE. Cationic triarylmethane dyes (TAM +)s which are used as colorants in industry and as frequent tools and reagents in analytical, cell biological and biomedical research have been recently characterized as reversible inhibitors of human butyrylcholinesterase. In this study, the inhibitory effects of two TAM +s, malachite green (MG) and methyl green (MeG) on five human BChE mutants (A277V, P285L, H77L, A328F and F329A) were studied spectrophotometrically at 25 °C in 50 mM MOPS buffer pH 8, using butyrylthiocholine as substrate. The kinetic results obtained with mutant enzymes were compared to those obtained with recombinant wild type BChE. MG and MeG were found to act as competitive/linear mixed inhibitors of recombinant wild type BChE and all BChE mutants except the F329A mutant. Both dyes caused complex nonlinear inhibition of F329A mutant, pointing to multisite binding. K i values for MG and MeG, estimated by nonlinear regression analysis, were 3.8 and 27 μM, respectively, as compared to the 50- to 150-fold lower values observed with recombinant wild type BChE. The observed significant differences in kinetic pattern and K i values between recombinant wild type BChE and F329A mutant suggest that phenylalanine at position 329 in human BChE is a critical residue in MG and MeG binding to enzyme.