title: Multi-step analysis as a tool for kinetic parameter estimation and mechanism discrimination in the reaction between tight-binding fasciculin 2 and electric eel acetylcholinesterase.
abstract: The mechanism of action of a potent peptidic inhibitor fasciculin 2 (Fas2) on electric eel acetylcholinesterase (eleelAChE) has been examined in a three-level analysis. Classical steps included equilibration experiments for the evaluation of high affinity binding constant and the existence of residual hydrolytic activity in a solution of completely Fas2 saturated enzyme. The two rate constants for the association (k(on)) and the dissociation (k(off)) of Fas2 with free enzyme were determined by the time course of residual enzyme activity measurements. In the third step, with a nonclassical progress curve analysis, we found that the Fas2-enzyme complex exhibited hydrolytic activity in a butyrylcholinesterase-like kinetics. The switch appears to be a consequence of steric obstruction, but also the consequence of subtle rapid conformational changes around catalytic site, upon slow single-step binding of large Fas2 molecule at the peripheral site. An unusual unilateral effect of bound Fas2 is reflected by acylation-independent association and dissociation rates and might indeed be due to inability of small acylation agent to influence the binding of a large opponent.
authors: Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia.
authors: Marko
authors: M
authors: Golicnik
authors: Jure
authors: J
authors: Stojan
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry*; D000122:Acetylthiocholine / Q000737:chemistry; D000818:Animals; D002800:Cholinesterase Inhibitors / Q000494:pharmacology*; D004546:Elapid Venoms / Q000494:pharmacology*; D004593:Electrophorus; D007700:Kinetics; D011485:Protein Binding; D012044:Regression Analysis
chemical_list: D002800:Cholinesterase Inhibitors; D004546:Elapid Venoms; D000122:Acetylthiocholine; C044937:fasciculin; D000110:Acetylcholinesterase