Microcalorimetric study of the inhibition of butyrylcholinesterase by carbamates.

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by three carbamates (eserine, neostigmine, and rivastigmine) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5). The kinetics of carbamylation was studied in the absence or presence of the substrate, butyrylcholine, using an extension of the model described by Stojan and coworkers (FEBS Lett. 440 (1998) 85-88). The model was fitted to the data by a nonlinear regression procedure using simulated annealing followed by Marquardt's method. The affinity of the carbamates for the free enzyme increased in the order neostigmine<eserine<rivastigmine, whereas the carbamylation rates followed an inverse order. In the case of rivastigmine, the results suggested that an acyl-enzyme-inhibitor complex could be formed either by reversible binding of the carbamate to the acyl-enzyme or by acylation of the enzyme-inhibitor complex. This work confirms the usefulness of microcalorimetry to study irreversible or progressive inhibitors and emphasizes the need to investigate the binding of such compounds to intermediate enzyme forms.