6-COUMARIN DIAZONIUM SALT - A SPECIFIC AFFINITY LABEL OF THE TORPEDO ACETYLCHOLINESTERASE PERIPHERAL SITE

A 6-coumarin diazonium salt was synthesized and tested on Torpedo acet ylcholinesterase as a site-directed irreversible probe for quaternary ammonium binding. The rate of the inactivation was examined as a funct ion of time, inhibitor concentration, and pH, which allowed the determ ination of the dissociation and the rate constants of this efficient a ffinity labeling process. Protection experiments using tetramethylammo nium, edrophonium, and propidium demonstrated that the labeling reacti on occurred exclusively at the peripheral quaternary ammonium binding site of the enzyme. This result was confirmed by the modification of p ropidium binding at the peripheral site after inactivation reaction, a s directly determined by fluorescence. Mutations of the likely labeled amino acid residues, Tyr(70) and Tyr(121), by histidine and phenylala nine indicated a predominant involvement of Tyr(70) over Tyr(121) in t he coupling reaction.