L-阿拉伯糖和D-半乳糖与大肠杆菌L-阿拉伯糖结合蛋白结合的热力学

Thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli.

作者信息

Fukada H, Sturtevant J M, Quiocho F A

出版信息

J Biol Chem. 1983 Nov 10;258(21):13193-8.

Abstract

The thermodynamics of the binding of L-arabinose and of D-galactose to the L-arabinose-binding protein of Escherichia coli have been studied by isothermal and scanning calorimetry. The binding reaction with arabinose is characterized by an enthalpy change of -15.3 +/- 0.5 kcal mol-1 at 25 degrees C, and a large decrease in apparent heat capacity, amounting to -0.44 +/- 0.05 kcal K-1 mol-1, which is constant over the temperature range 8 to 30 degrees C. Very similar results were obtained with D-galactose. These calorimetric results have been combined with binding constants determined by equilibrium dialysis (Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227-2233) to obtain free energy and entropy changes over the range 5 to 30 degrees C, and by extrapolation to 60 degrees C. The protein undergoes reversible unfolding on being heated with an increase in enthalpy at 53.5 degrees C of 151.8 +/- 1.1 kcal mol-1 (169.2 +/- 1.2 kcal mol-1 at 59.0 degrees C) and in apparent heat capacity of 3.16 +/- 0.07 kcal K-1 mol-1. In the presence of arabinose, the unfolding enthalpy is increased to 200.7 +/- 1.8 kcal mol-1 at 59.0 degrees C, the increase being due to the enthalpy of dissociation of the ligand which amounts to 31 kcal mol-1 at the unfolding temperature. The unfolding temperature is increased by the presence of excess arabinose or galactose, an effect which is due solely to displacement by the added ligand of the unfolding-dissociation equilibrium. The thermodynamic data are discussed in connection with the detailed structural information available for this system from x-ray crystallography (Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213-13217, and references cited therein).

摘要

通过等温滴定热分析和扫描量热法研究了L-阿拉伯糖和D-半乳糖与大肠杆菌L-阿拉伯糖结合蛋白的结合热力学。与阿拉伯糖的结合反应在25℃时的焓变为-15.3±0.5 kcal·mol⁻¹，表观热容大幅下降，达到-0.44±0.05 kcal·K⁻¹·mol⁻¹，在8至30℃的温度范围内保持恒定。用D-半乳糖得到了非常相似的结果。这些量热结果与通过平衡透析测定的结合常数（Clark, A. F., Gerken, T. A., and Hogg, R. W. (1982) Biochemistry 21, 2227 - 2233）相结合，以获得5至30℃范围内以及外推至60℃时的自由能和熵变。该蛋白质在加热时会发生可逆的去折叠，在53.5℃时焓增加151.8±1.1 kcal·mol⁻¹（在59.0℃时为169.2±1.2 kcal·mol⁻¹），表观热容增加3.16±0.07 kcal·K⁻¹·mol⁻¹。在阿拉伯糖存在下，59.0℃时去折叠焓增加到200.7±1.8 kcal·mol⁻¹，增加是由于配体解离的焓，在去折叠温度下为31 kcal·mol⁻¹。过量阿拉伯糖或半乳糖的存在会提高去折叠温度，这种效应完全是由于添加的配体取代了去折叠 - 解离平衡。结合X射线晶体学（Newcomer, M. E., Gilliland, G. L. and Quiocho, F. A. (1981) J. Biol. Chem. 256, 13213 - 13217，以及其中引用的参考文献）可获得的该系统详细结构信息，对热力学数据进行了讨论。