伴刀豆球蛋白A中金属离子取代对碳水化合物结合及热稳定性的影响。

Effect of metal ion substitutions in concanavalin A on the binding of carbohydrates and on thermal stability.

作者信息

Sanders J N, Chenoweth S A, Schwarz F P

机构信息

Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, Rockville, MD 20850, USA.

出版信息

J Inorg Biochem. 1998 May;70(2):71-82. doi: 10.1016/s0162-0134(98)00016-6.

DOI:10.1016/s0162-0134(98)00016-6

PMID:9666569

Abstract

Isothermal titration calorimetry (ITC) measurements were performed on the binding of alpha methyl-D-mannopyranoside, D-mannopyranose, alpha methyl-D-glucopyranoside, and D-glucopyranose (Glu) to cobalt, nickel, and cadmium substituted concanavalin A (Con A) derivatives at pH = 6.9 and at 25 degrees C. The metal substituted Con A derivatives consisted of Co2+, Ni2+, and Cd2+ substituted for the Mn2+ ion in the S1 site of Con A which is about 12.8 A away from the center of the carbohydrate binding site of Con A. The thermodynamic quantities determined from the ITC measurements were the same for most of the binding reactions indicating that the structure of the binding site in solution is the same for all the Con A derivatives in solution and that the presence of different 2+ metal ions in the S1 site has little effect on the binding reactions. Differential scanning calorimetry scans of solutions of the metal ion derivatives of Con A show that the thermodynamics of the unfolding transition for the cobalt and nickel substituted derivatives are the same as for Con A: they dissociate from tetramers into monomers as they unfold around 85 degrees C. The cadmium substituted Con A derivative, however, exhibits an additional transition around 93 degrees C which also appears following the addition of Cd2+ to the Con A solutions. This transition results from the unfolding of a species of Con A with Cd2+ substituted into a third binding site at the monomeric interface of the Con A tetramer. The higher stability of this species is not only exemplified by the higher thermal transition temperature but also by the lack of dissociation as it unfolds. Cd2+ is released from the S3 site upon decreasing the pH from 6.9 to 6.4. ITC measurements on the binding reaction of Cd2+ to Con A show that the binding enthalpy is 40.2 +/- 0.4 kJ mol-1 at 23.4 +/- 0.2 degrees C and the binding reaction exhibits a large heat capacity change of 1.43 +/- 0.41 kJ mol-1 K-1.

摘要

在pH = 6.9和25℃条件下，采用等温滴定量热法（ITC）测量了α-甲基-D-甘露吡喃糖苷、D-甘露吡喃糖、α-甲基-D-葡萄糖吡喃糖苷和D-葡萄糖（Glu）与钴、镍和镉取代的伴刀豆球蛋白A（Con A）衍生物的结合情况。金属取代的Con A衍生物是指在Con A的S1位点中，Co2+、Ni2+和Cd2+取代了Mn2+离子，该位点距离Con A碳水化合物结合位点中心约12.8 Å。从ITC测量中确定的热力学量对于大多数结合反应来说是相同的，这表明溶液中结合位点的结构对于溶液中所有Con A衍生物都是相同的，并且S1位点中不同二价金属离子的存在对结合反应影响很小。对Con A金属离子衍生物溶液进行的差示扫描量热法扫描表明，钴和镍取代衍生物的解折叠转变热力学与Con A相同：它们在约85℃解折叠时从四聚体解离成单体。然而，镉取代的Con A衍生物在约93℃表现出额外的转变，在向Con A溶液中加入Cd2+后也会出现这种转变。这种转变是由于Con A的一种物种在Con A四聚体的单体界面处的第三个结合位点被Cd2+取代后解折叠所致。该物种的更高稳定性不仅体现在更高的热转变温度上，还体现在解折叠时不解离。当pH从6.9降至6.4时，Cd2+从S3位点释放。对Cd2+与Con A结合反应的ITC测量表明，在23.4±0.2℃时结合焓为40.2±0.4 kJ mol-1，结合反应表现出1.43±0.41 kJ mol-1 K-1的大的热容变化。