Hedstrom L, Lin T Y, Fast W
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254, USA.
Biochemistry. 1996 Apr 9;35(14):4515-23. doi: 10.1021/bi951928k.
Trypsinogen is converted to trypsin by the removal of a peptide from the N terminus, which permits formation of a salt bridge between the new N-terminal Ile (residue 16) and Asp194. Formation of this salt bridge triggers a conformational change in the "activation domain" of trypsin, creating the S1 binding site and oxyanion hole. Thus, the activation of trypsinogen appears to represent an example of protein folding driven by electrostatic interactions. The following trypsin mutants have been constructed to explore this problem: Asp194Asn, Ile16Val, Ile16Ala, and Ile16Gly. The bovine pancreatic trypsin inhibitor (BPTI), benzamidine, and leupeptin affinities and activity and pH-rate profiles of these mutants have been measured. The changes in BPTI and benzamidine affinity measure destabilization of the activation domain. These experiments indicate that hydrophobic interactions of the Ile16 side chain provide 5 kcal/mol of stabilization energy to the activation domain while the salt bridge accounts for 3 kcal/mol. Thus, hydrophobic interactions provide the majority of stabilization energy for the trypsinogen to trypsin conversion. The pH-rate profiles of I16A and I16G are significantly different than the pH-rate profile of trypsin, further confirming that the activation domain has been destabilized. Moreover, these mutations decrease kcat/Km and leupeptin affinity in parallel with the decrease in stability of the activation domain. Acylation is selectively decreased, while substrate binding and deacylation are not affected. Together these observations indicate that the stability of protein structure is an important component of transition state stabilization in enzyme catalysis. These results also suggest that active zymogens can be created without providing a counterion for Asp194, and thus have important implications for the elucidation of the structural features which account for the zymogen activity of tissue plasminogen activator and urokinase.
胰蛋白酶原通过从N端去除一个肽段而转化为胰蛋白酶，这使得新的N端异亮氨酸（残基16）与天冬氨酸194之间形成盐桥。该盐桥的形成触发了胰蛋白酶“激活结构域”的构象变化，产生了S1结合位点和氧阴离子洞。因此，胰蛋白酶原的激活似乎代表了由静电相互作用驱动的蛋白质折叠的一个例子。构建了以下胰蛋白酶突变体以探究这个问题：天冬氨酸194→天冬酰胺、异亮氨酸16→缬氨酸、异亮氨酸16→丙氨酸和异亮氨酸16→甘氨酸。已测量了这些突变体的牛胰蛋白酶抑制剂（BPTI）、苯甲脒和亮抑酶肽亲和力以及活性和pH-速率曲线。BPTI和苯甲脒亲和力的变化衡量了激活结构域的不稳定。这些实验表明，异亮氨酸16侧链的疏水相互作用为激活结构域提供了5千卡/摩尔的稳定能，而盐桥贡献了3千卡/摩尔。因此，疏水相互作用为胰蛋白酶原向胰蛋白酶的转化提供了大部分稳定能。I16A和I16G的pH-速率曲线与胰蛋白酶的pH-速率曲线显著不同，进一步证实激活结构域已不稳定。此外，这些突变与激活结构域稳定性的降低平行地降低了kcat/Km和亮抑酶肽亲和力。酰化作用选择性降低，而底物结合和去酰化不受影响。这些观察结果共同表明，蛋白质结构的稳定性是酶催化中过渡态稳定的一个重要组成部分。这些结果还表明，无需为天冬氨酸194提供抗衡离子就可以产生有活性的酶原，因此对于阐明组织纤溶酶原激活剂和尿激酶的酶原活性的结构特征具有重要意义。
