The inactivation process of native (N) human butyrylcholinesterase (BuChE) by pressure and/or heat was found to be multi-step. It led to irreversible formation of an active intermediate (I) state and a denatured state. This series-inactivation process was described by expanding the Lumry–Eyring [Lumry, R. and Eyring, H. (1954) J. Phys. Chem. 58, 110–120] model. The intermediate state (I) was found to have a Km identical with that of the native state and a turnover rate (kcat) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (kcat/Km) of I can be explained by a conformational change in the active-site gorge and/or restructuring of the water-molecule network in the active-site pocket, making the catalytic steps faster. However, a pressure/heat-induced covalent modification of native BuChE, affecting the catalytic machinery, cannot be ruled out. The inactivation process of BuChE induced by the combined action of pressure and heat was found to continue after interruption of pressure/temperature treatment. This secondary inactivation process was termed ‘remnant inactivation’. We hypothesized that N and I were in equilibrium with populated metastable N′ and I′ states. The N′ and I′ states can either return to the active forms, N and I, or develop into inactive forms, N′in and I′in. Both active N′ and I′ intermediate states displayed different rates of remnant inactivation depending on the pressure and temperature pretreatments and on the storage temperature. A first-order deactivation model describing the kinetics of the remnant inactivation of BuChE is proposed.
Abbreviations used: BuChE, butyrylcholinesterase; AChE, acetylcholinesterase; ChE, cholinesterase; BuSCh, butyrylthiocholine iodide; CPO, chlorpyrifos-oxon; P/T, pressure/temperature; N, native human butyrylcholinesterase; I, active intermediate-state butyrylcholinesterase; D, irreversiblydenatured-state inactive butyrylcholinesterase; P0, atmospheric pressure.
Present address: Nestec Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland.