Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: Effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin
Alanine point-mutations in the reactive region of bovine pancreatic trypsin inhibitor: Effects on the kinetics and thermodynamics of binding to beta-trypsin and alpha-chymotrypsin
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DOI：
10.1021/bi960515w
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发表时间：
1996-09-03
期刊：
影响因子：
2.9
通讯作者：
Anderson, S
中科院分区：
文献类型：
--
作者：
Castro, MJM;Anderson, S
为了关联模型大分子识别过程中的结构、动力学和热力学特征，分别用丙氨酸取代了牛胰蛋白酶抑制剂(BPTI)活性表面的氨基酸残基及其周围残基，并研究了点突变对BPTI对胰酶和糜蛋白酶抑制的动力学和热力学的影响。共产生15个丙氨酸突变体。大多数BPTI突变体表现出与野生型蛋白相似的结合亲和力。圆二色谱和热稳定性测量显示，主要特异性位点(P1)突变体和那些似乎具有非局部结构扰动的突变体是例外。P1位的突变使胰酶和胰凝乳酶的结合自由能分别降低了10和1.8千卡摩尔(-1)。结合亲和力的损失几乎完全由解离速率常数的增加决定。然而，P1突变体Lys-15-Ala与胰酶的结合速度也显著降低(>；200倍)。通过测量凝乳酶与野生型抑制剂及其丙氨酸突变体结合的结合热，可以确定结合自由能与结合能和熵变化的相对贡献。在几种情况下观察到了这两个参数的补偿性变化，这归因于结合界面的去溶解效应。
In an effort to relate structural, kinetic, and thermodynamic features in a model macromolecular recognition process, the amino acid residues in the reactive surface of bovine pancreatic trypsin inhibitor (BPTI) and surrounding residues were substituted individually by alanine, and the effects of the point-mutations on the kinetics and thermodynamics of inhibition by BPTI toward trypsin and chymotrypsin were investigated. Fifteen alanine mutants were produced. The majority of the BPTI mutants exhibited a binding affinity similar to that of the wild-type protein. The exceptions were the primary specificity site (P1) mutant and those mutants that seem to have nonlocal perturbations of structure, as revealed by circular dichroism and thermostability measurements. The mutation at the P1 site caused a reduction in the binding free energy of 10 and 1.8 kcal mol(-1) for trypsin and chymotrypsin, respectively. The losses in binding affinity were determined almost exclusively by an increase in the dissociation rate constant. However, the rate of association of the P1 mutant, Lys-15-Ala, with trypsin was also drastically reduced (>200-fold). Calorimetric measurements of the heats of binding for the association of chymotrypsin with the wild-type inhibitor and its alanine mutants allowed determination of the relative contributions of the changes in enthalpy and entropy to the free energy of binding. Compensatory changes in the two parameters were observed in several cases, which were attributed to desolvation effects at the binding interface.