Approaches for improved identification of mechanisms of enzyme inactivation

Abstract

This publication reviews the results of the authors’ studies dealing with the identification of mechanisms of enzyme inactivation. It presents both a critical assessment of traditional approaches of evaluation of enzyme inactivation kinetics from uniresponse experiments as well as the procedures of better design and evaluation of experiments which improve the analysis of inactivation mechanism and its corresponding kinetics. These methods are based on the simultaneous evaluation of inactivation experiments carried out at several temperatures or pHs. The inactivation mechanism is thus evaluated from the data set containing larger amount of information compared to a single inactivation curve when the influence of the factor of variation is unequivocally defined, for example, by the Arrhenius equation in the case of temperature or protonation and deprotonation equilibrium constants in the case of pH. It is demonstrated furthermore that the complexity of the mechanism of the thermal inactivation of free enzyme can be enhanced by utilising the combination of selective immobilization and multitemperature evaluation when the immobilization prevents some reactions in the ordinary inactivation pathway to occur. All methods are illustrated on case studies using yeast invertase.