title: Mechanism and kinetics of inactivation at 40-70 degrees C of the extracellular proteinase from Pseudomonas fluorescens 22F.
abstract: HPLC size exclusion chromatography experiments showed that during inactivation at 40-70 degrees C of the extracellular proteinase from Pseudomonas fluorescens 22F small molecular mass fragments were formed, indicating that autoproteolysis was at least one of the major causes of inactivation. The formation of small molecular mass fragments and the reaction order indicated that intermolecular autoproteolysis was more likely than intramolecular autodigestion. This was confirmed by computer simulations. The rate constants and the activation enthalpy (delta H++) and entropy (delta S++) for the reactions of the intermolecular autoproteolysis model were derived from computer simulations. delta H++ and delta S++ of the unfolding reaction were 504 kJ mol-1 and 1252 J mol K-1 respectively. delta H++ and delta S++ of the refolding reaction were strongly temperature dependent. The estimates for the enthalpy (delta H0) and entropy (delta S0) difference between the folded and unfolded state as derived from the reaction rate constants of unfolding and refolding were subject to large deviations, owing to accumulation of errors in the estimation of the kinetic characteristics.
authors.affiliation: Wageningen Agricultural University, Department of Food Science, The Netherlands.
authors.forename: E P
authors.initials: EP
authors.lastname: Schokker
authors.forename: M A
authors.initials: MA
authors.lastname: van Boekel
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D019277:Entropy; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008892:Milk / Q000382:microbiology; D011489:Protein Denaturation; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000302:isolation & purification; D013816:Thermodynamics
chemical_list: D010450:Endopeptidases