Edge V, Allewell N M, Sturtevant J M
Biochemistry. 1985 Oct 8;24(21):5899-906. doi: 10.1021/bi00342a032.
The thermal denaturation of the catalytic (c3) and regulatory (r2) subunits of Escherichia coli aspartate transcarbamoylase (c6r6) in the absence and presence of various ligands has been studied by means of highly sensitive differential scanning calorimetry. The denaturation of both types of subunit is irreversible as judged by the facts that the proteins coagulate when heated and that no endotherm is observed when previously scanned protein is rescanned. Despite this apparent irreversibility, there is empirical justification for analyzing the calorimetric data in terms of equilibrium thermodynamics as embodied in the van't Hoff equation. The observed curves of excess apparent specific heat vs. temperature are asymmetric and can be expressed within experimental uncertainty as the sums of sequential two-state steps, a minimum of two steps being required for r2 and three for c3. As previously reported [Vickers, K. P., Donovan, J. W., & Schachman, H. K. (1978) J. Biol. Chem. 253, 8493-8498], the addition of the effectors ATP and CTP raises the denaturation temperature of r2 and lowers that of c3 while the addition of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate raises the denaturation temperature of c3 and lowers that of r2. These effects vary with ligand concentration in the manner expected from the van't Hoff equation, indicating that they are simply manifestations of Le Chatelier's principle rather than being due to "stabilization" or "destabilization" of the proteins. The denaturational enthalpy is increased in those cases of ligand binding in which the denaturation temperature is increased, because of the contribution from the enthalpy of dissociation of the ligand.
利用高灵敏度差示扫描量热法研究了大肠杆菌天冬氨酸转氨甲酰酶（c6r6）的催化亚基（c3）和调节亚基（r2）在有无各种配体存在下的热变性。根据蛋白质加热时会凝固以及对先前扫描过的蛋白质再次扫描时未观察到吸热峰这两个事实判断，这两种亚基的变性都是不可逆的。尽管存在这种明显的不可逆性，但根据范特霍夫方程所体现的平衡热力学来分析量热数据仍有经验依据。观察到的过量表观比热与温度的曲线是不对称的，并且在实验误差范围内可以表示为连续的两态步骤的总和，r2至少需要两个步骤，c3需要三个步骤。如先前报道[Vickers, K. P., Donovan, J. W., & Schachman, H. K. (1978) J. Biol. Chem. 253, 8493 - 8498]，效应物ATP和CTP的加入会提高r2的变性温度并降低c3的变性温度，而双底物类似物N -（膦酰基乙酰基）-L -天冬氨酸的加入会提高c3的变性温度并降低r2的变性温度。这些效应随配体浓度的变化符合范特霍夫方程预期的方式，表明它们仅仅是勒夏特列原理的体现，而非由于蛋白质的“稳定化”或“去稳定化”。在配体结合导致变性温度升高的情况下，变性焓会增加，这是由于配体解离焓的贡献。
