Johnson P E, Creagh A L, Brun E, Joe K, Tomme P, Haynes C A, McIntosh L P
Protein Engineering Network of Centres of Excellence, The University of British Columbia, Vancouver, Canada.
Biochemistry. 1998 Sep 15;37(37):12772-81. doi: 10.1021/bi980978x.
The interaction of the N-terminal cellulose-binding domain, CBDN1, from Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investigated using NMR spectroscopy and calorimetry. CBDN1 binds a single calcium ion with an equilibrium association constant of approximately 10(5) M-1 at 35 degreesC and pH 6.0. Binding is exothermic (-42 +/- 2 kJ mol-1) under these conditions and is accompanied by a small negative change in heat capacity (DeltaCp = -0.41 +/- 0.16 kJ mol-1 K-1). From an NMR line shape analysis, the rate constants for calcium association and dissociation were found to be (5 +/- 2) x 10(7) s-1 M-1 and (4.5 +/- 0.6) x 10(2) s-1, respectively. The rapid association kinetics indicate that the calcium-binding site on CBDN1 is accessible and, to the first approximation, preformed. Based on patterns of chemical shift perturbations, and structural comparisons with the Bacillus sp. 1, 3-1,4-beta-glucanases, the backbone carbonyl oxygens of Thr8, Gly30, and Asp142 and a side chain carboxyl oxygen of Asp142 are postulated to form the calcium-binding site of CBDN1. Consistent with the calcium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosaccharide-binding cleft. The midpoint denaturation temperature of CBDN1 is increased by approximately 8 degreesC at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.
利用核磁共振光谱法和量热法研究了纤维单胞菌β-1,4-葡聚糖酶CenC的N端纤维素结合结构域CBDN1与钙的相互作用。在35℃和pH 6.0条件下，CBDN1结合单个钙离子，平衡缔合常数约为10⁵ M⁻¹。在此条件下，结合是放热的（-42±2 kJ mol⁻¹），并伴随着热容的小负变化（ΔCp = -0.41±0.16 kJ mol⁻¹ K⁻¹）。通过核磁共振线形分析，发现钙缔合和解离的速率常数分别为(5±2)×10⁷ s⁻¹ M⁻¹和(4.5±0.6)×10² s⁻¹。快速缔合动力学表明，CBDN1上的钙结合位点是可及的，并且初步看来是预先形成的。基于化学位移扰动模式以及与芽孢杆菌属1,3-1,4-β-葡聚糖酶的结构比较，推测Thr8、Gly30和Asp142的主链羰基氧以及Asp142的侧链羧基氧形成了CBDN1的钙结合位点。与CBDN1对纤维五糖的钙非依赖性亲和力一致，这个暴露的位点位于CBDN1与形成寡糖结合裂隙的面相对的面上。在pH 6.0且存在饱和量钙的情况下，CBDN1的中点变性温度升高了约8℃，证实金属离子结合在热力学上与天然态稳定性相关。
