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 acetylcholinesterase as a site-directed irreversible probe for quaternary ammonium binding. The rate of the inactivation was examined as a function of time, inhibitor concentration, and pH, which allowed the determination of the dissociation and the rate constants of this efficient affinity labeling process. Protection experiments using tetramethylammonium, edrophonium, and propidium demonstrated that the labeling reaction occurred exclusively at the peripheral quaternary ammonium binding site of the enzyme. This result was confirmed by the modification of propidium binding at the peripheral site after inactivation reaction, as directly determined by fluorescence. Mutations of the likely labeled amino acid residues, Tyr70 and Tyr121, by histidine and phenylalanine indicated a predominant involvement of Tyr70 over Tyr121 in the coupling reaction.
Schalk
I
I
Laboratoire de Chimie Bio-organique, URA 1386 CNRS, Faculté de Pharmacie, Université Louis Pasteur Strasbourg, Illkirch, France.
Ehret-Sabatier
L
L
Le Feuvre
Y
Y
Bon
S
S
Massoulie
J
J
Goeldner
M
M
D000110:Acetylcholinesterase / Q000235:genetics / Q000378:metabolism*; D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D003374:Coumarins; D004789:Enzyme Activation; D007700:Kinetics; D009154:Mutation; D012680:Sensitivity and Specificity; D014101:Torpedo; D014443:Tyrosine / Q000378:metabolism
C097271:6-coumarin diazonium salt; D000345:Affinity Labels; D003374:Coumarins; D014443:Tyrosine; D000110:Acetylcholinesterase