Thermodynamic analysis of the stabilisation 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 mitochondrial malate dehydrogenase (phm-MDH) and maize leaf phosphoenolpyruvate carboxylase (ml-PEPC) in the presence of various cosolvents, the denaturation kinetics as a function of temperature have been determined based on Arrhenius plots derived from transition state theory analysis 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 concentrations of various salts of monovalent and polyvalent anions, 1 M amino acids or 1 M sucrose and 6.1 M glycerol. Similarly, Arrhenius plot 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 the phm-MDH in the presence of all cosolvents and for the ml-PEPC in the presence of 2.5 M NaOAc, but not in the presence of 0.8 M sodium glutamate. A significant temperature-dependent effect dominated the inactivation of phm-MDH and ml-PEPC at elevated temperatures (e.g., > or = 45 degrees C), whilst the inactivation of these enzymes over a lower temperature range (< or = 25 degrees C) was dominated by temperature-independent phenomenon. The corresponding thermodynamic activation parameters (deltaG++, deltaH++ and deltaS++) associated with the transition state complexes involved in the inactivation of phm-MDH and ml-PEPC in the presence of the various cosolvents have been determined. The results indicate that the transition states associated with the inactivation of these two enzymes at elevated temperatures are characterised by large, positive enthalpic and entropic changes. In contrast, the inactivation process observed for phm-MDH at low temperatures in the presence 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 inactivation can occur with these two multimeric enzymes depending on the selected temperature range and the type of cosolvent. The relationship of these results to stabilisation models for phm-MDH and ml-PEPC in the presence of various cosolvents, as well as the application of Arrhenius plot data to extrapolate the long term solution stability of these enzymes at lower temperatures from the pseudo-first order rate constants of inactivation experimentally derived over a range of temperatures, are discussed.
Jensen
W A
WA
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia.
Armstrong
J M
JM
De Giorgio
J
J
Hearn
M T
MT
D000438:Alcohols / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D008291:Malate Dehydrogenase / Q000737:chemistry*; D008929:Mitochondria, Heart / Q000201:enzymology*; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry*; D012492:Salts / Q000737:chemistry; D012997:Solvents; D013552:Swine; D013696:Temperature; D013816:Thermodynamics; D003313:Zea mays / Q000201:enzymology*
D000438:Alcohols; D000596:Amino Acids; D012492:Salts; D012997:Solvents; D008291:Malate Dehydrogenase; D010730:Phosphoenolpyruvate Carboxylase