Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process

Abstract

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 K(m) identical with that of the native state and a turnover rate (k(cat)) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (k(cat)/K(m)) 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.

Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence

“…A transient activation phase was reported for the Co 2+ -PTE at pH 9.4 [13]. Transitory increase in activity during thermal inactivation was also reported for other enzymes [35][36][37][38]. Finally, Zn 2+ -PTE appeared to be the most thermostable enzyme and Cd 2+ -PTE the least ( Figure 5).…”

Effects of Soman Inhibition and of Structural Differences on Cholinesterase Molecular Dynamics: A Neutron Scattering Study

“…For HuBChE, it supports the notion that its denaturation is a concerted process, involving a large number of amino acid residues. Indeed, several lines of evidence indicate that both pressure-induced and thermal denaturation of HuBChE are multistep processes that are initiated by penetration of water into the active-site gorge, leading to protein swelling and, eventually, unfolding (18,53,54).…”

Anti-Inflammatory and Antioxidant Properties of Casein Hydrolysate Produced Using High Hydrostatic Pressure Combined with Proteolytic Enzymes

“…Therefore, high hydrostatic pressure combined with enzymatic hydrolysis (HHP-EH) is an eco-friendly process compared to enzymatic hydrolysis under atmospheric pressure (AP-EH), because it uses a lower amount of enzymes and solvents. Several studies have reported the use of extremely high-pressure (400–800 MPa) as a pre-treatment before enzymatic hydrolysis [ 12 , 15 , 16 ], which may result in unacceptable protein denaturation. Previously, HHP-EH was used to obtain bioactive peptides from β-lactoglobulin [ 17 ], ovalbumin [ 18 ], chickpea protein [ 19 ], and pinto bean protein [ 20 ].…”