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


BİBEROĞLU K. , TACAL Ö. , Akbulut H.

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, vol.511, pp.64-68, 2011 (Journal Indexed in SCI) identifier identifier identifier

  • Publication Type: Article / Article
  • Volume: 511
  • Publication Date: 2011
  • Doi Number: 10.1016/j.abb.2011.04.007
  • Title of Journal : ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
  • Page Numbers: pp.64-68
  • Keywords: Human BChE, Site-directed mutagenesis, Malachite green, Methyl green, Cholinesterase inhibition, HUMAN PLASMA CHOLINESTERASE, AMINO-ACID-RESIDUES, MALACHITE GREEN, ACETYLCHOLINESTERASE, INHIBITION, TRIPHENYLMETHANE, PHENOXAZINE, RATS

Abstract

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 degrees 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 mu M, 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. (C) 2011 Elsevier Inc. All rights reserved.