Jensen W A, Armstrong J M, De Giorgio J, Hearn M T
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia.
Biochim Biophys Acta. 1997 Apr 4;1338(2):186-98. doi: 10.1016/s0167-4838(96)00198-7.
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.
作为我们对猪心线粒体苹果酸脱氢酶（phm-MDH）和玉米叶片磷酸烯醇式丙酮酸羧化酶（ml-PEPC）在各种共溶剂存在下失活情况研究的一部分，基于从3.5℃至65℃温度范围内过渡态理论分析得出的阿伦尼乌斯图，测定了变性动力学随温度的变化。phm-MDH的实验数据是在1 M浓度的各种单价和多价阴离子盐、1 M氨基酸或1 M蔗糖以及6.1 M甘油存在的情况下获得的。同样，ml-PEPC的阿伦尼乌斯图数据是在2.5 M醋酸钠和0.8 M谷氨酸钠存在的情况下获得的。在所有共溶剂存在下的phm-MDH以及在2.5 M醋酸钠存在下的ml-PEPC中，确定了对应于高失活焓值和低失活焓值的不同失活状态，但在0.8 M谷氨酸钠存在下未观察到。在较高温度（例如，≥45℃）时，显著的温度依赖性效应主导了phm-MDH和ml-PEPC的失活，而在较低温度范围（≤25℃）内这些酶的失活则由与温度无关的现象主导。已经确定了与在各种共溶剂存在下phm-MDH和ml-PEPC失活过程中涉及的过渡态复合物相关的相应热力学活化参数（ΔG++、ΔH++和ΔS++）。结果表明，在较高温度下与这两种酶失活相关的过渡态具有较大的正焓变和熵变特征。相比之下，在各种共溶剂存在下phm-MDH在低温下观察到的失活过程的特征是有较大的负熵贡献和较小的正焓贡献。本研究获得的结果表明，根据所选温度范围和共溶剂类型，这两种多聚体酶可能存在不止一种失活机制。讨论了这些结果与phm-MDH和ml-PEPC在各种共溶剂存在下的稳定化模型的关系，并讨论了如何应用阿伦尼乌斯图数据，根据在一系列温度下实验得出的伪一级失活速率常数来推断这些酶在较低温度下的长期溶液稳定性。
