Twomey C M, Doonan S
Department of Biochemistry, University College, Cork, Ireland.
Biochim Biophys Acta. 1997 Sep 26;1342(1):37-44. doi: 10.1016/s0167-4838(97)00075-7.
Rates of irreversible thermal inactivation of cytosolic and mitochondrial aspartate aminotransferases were measured over a large temperature range. Inactivation occurred by different kinetic pathways at high and low temperature with a transition point at about 60 degrees C. This suggests that the isoenzymes exist in different conformations above and below that temperature. Discontinuities in plots of ln(Vmax) against 1/T provided confirmatory evidence for this hypothesis. Activation parameters (deltaH and deltaS) for the thermal inactivation process were calculated in the high and low temperature ranges. At high temperature the greater rate of inactivation of the mitochondrial isoenzyme is determined largely by a high value of deltaS. This more than compensates for the fact that the deltaH is also greater for the mitochondrial isoenzyme indicative of greater intramolecular stabilising interactions compared with the cytosolic form. Thus the relative rates of inactivation are determined by the nature of the transition states rather than by intramolecular interactions in the folded proteins. At lower temperatures the kinetic stabilities of the isoenzymes reverse with the mitochondrial isoenzyme inactivating more slowly. This is largely because of a considerably smaller deltaS at low temperature which no longer compensates for the greater deltaH compared with the cytosolic isoenzyme.
在较大温度范围内测定了胞质和线粒体天冬氨酸氨基转移酶不可逆热失活的速率。在高温和低温下，失活通过不同的动力学途径发生，转变点约为60摄氏度。这表明同工酶在该温度以上和以下以不同构象存在。ln(Vmax)对1/T的图中的不连续性为该假设提供了确证。计算了高温和低温范围内热失活过程的活化参数（ΔH和ΔS）。在高温下，线粒体同工酶较高的失活速率主要由较高的ΔS值决定。这足以弥补线粒体同工酶的ΔH也较高这一事实，这表明与胞质形式相比，分子内稳定相互作用更强。因此，失活的相对速率由过渡态的性质决定，而不是由折叠蛋白中的分子内相互作用决定。在较低温度下，同工酶的动力学稳定性相反，线粒体同工酶失活更慢。这主要是因为低温下的ΔS小得多，不再能弥补与胞质同工酶相比更大的ΔH。
