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.