THERMODYNAMIC ANALYSIS OF THE STABILIZATION OF PIG-HEART MITOCHONDRIAL MALATE-DEHYDROGENASE AND MAIZE LEAF PHOSPHOENOLPYRUVATE CARBOXYLASE BY DIFFERENT SALTS, AMINO-ACIDS AND POLYOLS

As part of our investigations into the inactivation of pig heart mitoc hondrial malate dehydrogenase (phm-MDH) and maize leaf phospho enolpyr uvate carboxylase (ml-PEPC) in the presence of various cosolvents, the denaturation kinetics as a function of temperature have been determin ed based on Arrhenius plots derived from transition state theory analy sis over the temperature range from 3.5 degrees C to 65 degrees C. The experimental data for phm-MDH were obtained in the presence of 1 M co ncentrations of various salts of monovalent and polyvalent anions, 1 M amino acids or 1 M sucrose and 6.1 M glycerol. Similarly, Arrhenius p lot data were obtained for ml-PEPC in the presence of 2.5 M NaOAc and 0.8 M sodium glutamate. Distinct regimes of inactivation corresponding to high and low values of inactivation enthalpy were identified for t he phm-MDH in the presence of all cosolvents and for the ml-PEPC in th e presence of 2.5 M NaOAc, but not in the presence of 0.8 M sodium glu tamate. A significant temperature-dependent effect dominated the inact ivation of phm-MDH and ml-PEPC at elevated temperatures (e.g., greater than or equal to 45 degrees C), whilst the inactivation of these enzy mes over a lower temperature range (less than or equal to 25 degrees C ) was dominated by temperature-independent phenomenon. The correspondi ng thermodynamic activation parameters (Delta G(double dagger), Delta H-double dagger and Delta S-double dagger) associated with the transit ion state complexes involved in the inactivation of phm-MDH and ml-PEP C in the presence of the various cosolvents have been determined. The results indicate that the transition states associated with the inacti vation of these two enzymes at elevated temperatures are characterised by large, positive enthalpic and entropic changes. In contrast, the i nactivation process observed for phm-MDH at low temperatures in the pr esence of various cosolvents was marked by a large, negative entropic contribution and a small, positive enthalpic contribution. The results obtained in this study indicate that more than one mechanism of inact ivation can occur with these two multimeric enzymes depending on the s elected temperature range and the type of cosolvent. The relationship of these results to stabilisation models for phm-MDH and ml-PEPC in th e presence of various cosolvents, as well as the application of Arrhen ius plot data to extrapolate the long term solution stability of these enzymes at lower temperatures from the pseudo-first order rate consta nts of inactivation experimentally derived over a range of temperature s, are discussed.