Kinetics of heat inactivation of phenyl valerate hydrolases from hen and rat brain

Abstract

Heat inactivation was studied at 45, 50, 55, and 60° for all of the phenyl valerate hydrolases (PVase), including neurotoxic esterase (NTE) and inhibitor-resistant esterase (IRE), in homogenates of hen or rat brain or in preparations of hen brain microsomal membranes. Hen and rat brain homogenates were prepared in buffer (50 mM Tris/0.20 mM EDTA, pH 8.00, at 25°). Hen brain microsomes were suspended either in buffer or in aqueous dimethyl sulfoxide (DMSO, 40%, w v ), or solubilized either in aqueous Triton X-100 (0.10%, w v ) or in 40% ( w v ) DMSO. Enzyme activities were measured at 37° using phenyl valerate as substrate. Each enzyme activity in all of the preparations exhibited biphasic heat inactivation kinetics. Apparent rate constants were calculated for the fast ( k f) and slow ( k s) reactions, along with the relative amounts of activity in each component ( A f, A s) expressed as percentages of the total activity. For a given preparation and temperature, respective values of k f or k s were similar for PVase, NTE, and IRE, with a mean k f k s , ratio of 52 across all preparations. A f and A s, were a func of temperature. Mean values of the apparent activation energies ( E a) for all activities and preparations were 44 and 25 kcal/mol for the fast and slow inactivation reactions respectively. These results indicate that all phenyl valerate hydrolases in hen and rat brain undergo a common heat-induced structural change leading to loss of enzymic activity.