Abou-Hachem Maher, Karlsson Eva Nordberg, Simpson Peter J, Linse Sara, Sellers Peter, Williamson Michael P, Jamieson Stuart J, Gilbert Harry J, Bolam David N, Holst Olle
Department of Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Biochemistry. 2002 May 7;41(18):5720-9. doi: 10.1021/bi012094a.
Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
利用量热法、核磁共振（NMR）、荧光法和吸收光谱法，研究了来自嗜热栖热菌的木聚糖酶10A（Xyn10A）的碳水化合物结合模块CBM4-2与钙的结合情况。CBM4-2结合两个钙离子，一个具有中等亲和力，另一个具有极高亲和力。中等亲和力位点的缔合常数为(1.3±0.3)×10⁵ M⁻¹，结合焓ΔH(a)为-9.3±0.4 kJ·mol⁻¹，而高亲和力位点的缔合常数约为10¹⁰ M⁻¹，结合焓ΔH(a)为-40.5±0.5 kJ·mol⁻¹。结合位点的位置已通过核磁共振和结构同源性确定，并通过定点诱变进行了验证。高亲和力位点由E11和D160的侧链以及E52和K55的主链羰基组成，而中等亲和力位点由D29的侧链以及L21、A22、V25和W28的主链羰基组成。高亲和力位点的位置类似于CBM4结构和最近的CBM22结构中的钙位点。在pH 7.5时，钙的结合使蛋白质的解折叠温度（T(m)）提高了约23℃。未观察到结合亲和力与T(m)变化之间的相关性，因为两个钙离子对解折叠温度升高的贡献几乎相同。
