Kinetic characterization of all steps of the interaction between acetylcholinesterase and eserine.
The three-step carbamylenzyme mechanism of the action of eserine on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) has been known for a long time, but its complete kinetic characterization has never been done. Some of our investigations indicated that the determination of missing kinetic parameters should include the inspection of the enzyme-eserine interaction in a very wide range of eserine concentrations. Therefore, the activity of acetylcholinesterase as a function of time in the presence of low concentrations of eserine comparable to the enzyme concentration was followed. The reaction mechanism was analysed by fitting numerically integrated differential equations that describe the time dependences of all reactants and reaction intermediates to these data. Additionally, the progress curve measurements at higher eserine concentrations were carried out on a stopped-flow apparatus. The corresponding progress curve equations were derived and the kinetic parameters evaluated by non-linear regression treatment. The complex analysis confirmed the three-step mechanism. The values of the constants showed that the very high affinity of eserine for binding into the active centre of the enzyme is not so much a consequence of the fast initial complex formation but rather a consequence of its slow dissociation. The subsequent covalent bonding of eserine is also slow, but faster than the dissociation of the initial complex. In this manner, the decarbamoylation is the only process responsible for the reactivation of acetylcholinesterase after removal of eserine.
Stojan
J
J
Institute of Biochemistry, Medical Faculty, University of Ljubljana, Slovenia.
Zorko
M
M
D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002800:Cholinesterase Inhibitors / Q000378:metabolism*; D004555:Electric Fish; D007700:Kinetics; D008956:Models, Chemical; D010830:Physostigmine / Q000378:metabolism*; D013223:Statistics as Topic
D002800:Cholinesterase Inhibitors; D010830:Physostigmine; D000110:Acetylcholinesterase