p-Butyroxybenzenediazonium fluoroborate, substrate of acetylcholinesterase and butyrylcholinesterase, discriminates between the two enzymes by a specific affinity labelling.
p-Butyroxybenzenediazonium fluoroborate 1 was shown to be a substrate of both acetylcholinesterase (AcChE) and butyrylcholinesterase (BuChE) with Michaelis constants of 6.10(-5) M and 1.3. 10(-4)M, respectively. Upon incubation in the dark, 1 was able to discriminate between the two enzymes AcChE was efficiently inactivated in a time-dependent manner while BuChE remained unaffected. Kinetic analysis of the inactivation of AcChE (i) by various concentrations of 1 indicated that it behaves as an affinity label, (ii) at three different pH levels suggested that the pKa of the labelled residue was higher than 7 and (iii) in the presence of different selective ligands for either the active site (edrophonium) or the peripheral site (propidium) indicated that 1 alkylated the active site rather than the peripheral one. Differences of reactivity between AcChE and BuChE suggest a different positioning and/or a different chemical environment of the substrate within two active sites.
Ehret-Sabatier
L
L
Laboratoire de Chimie Bioorganigue, CNRS URA 1386, Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France.
Goeldner
M P
MP
Hirth
C G
CG
D000110:Acetylcholinesterase / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000378:metabolism*; D003979:Diazonium Compounds / Q000378:metabolism*; D004491:Edrophonium / Q000378:metabolism; D004557:Electric Organ / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011419:Propidium / Q000378:metabolism; D013379:Substrate Specificity; D014101:Torpedo
D000345:Affinity Labels; D003979:Diazonium Compounds; C066838:4-butyroxybenzenediazonium fluoroborate; D011419:Propidium; D004491:Edrophonium; D000110:Acetylcholinesterase; D002091:Butyrylcholinesterase