CONTRIBUTION OF RESIDUES IN THE REACTIVE-SITE LOOP OF CHYMOTRYPSIN INHIBITOR-2 TO PROTEIN STABILITY AND ACTIVITY
CONTRIBUTION OF RESIDUES IN THE REACTIVE-SITE LOOP OF CHYMOTRYPSIN INHIBITOR-2 TO PROTEIN STABILITY AND ACTIVITY
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DOI：
10.1021/bi00250a042
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发表时间：
1994-11-22
期刊：
影响因子：
2.9
通讯作者：
FERSHT, AR
中科院分区：
文献类型：
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作者：
JACKSON, SE;FERSHT, AR
丝氨酸蛋白酶抑制剂胰凝乳蛋白酶抑制剂 2 活性位点环中的残基被认为在环稳定性和抑制活性中发挥重要作用，已通过定点诱变进行了研究。残基 58（野生型中的苏氨酸）和 60（野生型中的谷氨酸）位于易断键 (Met-59-Glu-60) 侧翼，并且在丝氨酸蛋白酶抑制剂的马铃薯抑制剂 I 家族中保守，发现残基 58（野生型中的苏氨酸）和 60（野生型中的谷氨酸）的取代对于蛋白质的整体稳定性具有一定的重要性（通过氯化胍诱导的变性测量），但对其抑制活性至关重要。 Thr-58 或 Glu-60 突变为丙氨酸会导致稳定性降低 0.7 +/- 0.1 kcal mol(-1)。这些值反映了 Thr-58 的羟基与 Glu-60 和 Arg-67 之间的氢键以及 Glu-60 与 Arg-62 和 Arg-65 之间的氢键和盐桥的损失。此外，还发现这些突变体对丝氨酸蛋白酶枯草杆菌蛋白酶 BPN' 的抑制剂要弱得多。对于野生型 T58A，抑制解离常数 K-i 为 (7.0 +/- 0.4、540 +/- 30 和 980 +/- 50) x 10(-13) M；和E60A，分别。此外，我们发现这些突变体只是枯草杆菌蛋白酶 BPN' 的临时抑制剂，与野生型不同。在很长一段时间内，我们观察到由于抑制剂的水解而抑制作用的逆转。此外，我们从T58AE60A和T58DE60A的双突变体循环分析中发现，游离抑制剂中残基58和60之间的相互作用能分别为0.5+/-0.2和0.4+/-0.1kcalmol(-1)（分别针对T58AE60A和T58DE60A测量），在与枯草杆菌蛋白酶BPN'的复合物中显着增加分别为 2.1 +/- 0.1 和 4.2 +/- 0.2 kcal mol(-1)。活性位点环内的氢键和盐桥网络是马铃薯抑制剂 I 家族抑制剂的特征，对于蛋白质的抑制活性至关重要。
Residues in the active site loop of the serine protease inhibitor, chymotrypsin inhibitor 2, thought to play an important role in loop stability and inhibitory activity, have been investigated by site-directed mutagenesis. Substitutions at residues 58 (threonine in wild type) and 60 (glutamic acid in wild type), which flank the scissile bond (Met-59-Glu-60) and are conserved among the potato inhibitor I family of serine protease inhibitors, are found to be of some importance in the global stability of the protein, as measured by guanidinium chloride-induced denaturation, but are essential for its inhibitory activity. Mutation of either Thr-58 or Glu-60 to alanine results in a decrease in stability of 0.7 +/- 0.1 kcal mol(-1). These values reflect the loss of hydrogen bonds between the hydroxyl group of Thr-58 with Glu-60 and Arg-67 and hydrogen bonds and a salt bridge between Glu-60 and Arg-62 and Arg-65. In addition, these mutants were found to be much weaker inhibitors of the serine protease subtilisin BPN'. The dissociation constants for inhibition, K-i, were found to be (7.0 +/- 0.4, 540 +/- 30, and 980 +/- 50) x 10(-13) M, for wild type, T58A; and E60A, respectively. Further, we find that these mutants are only temporary inhibitors of subtilisin BPN', unlike wild type. Over long time scales, we observe a reversal of inhibition because of hydrolysis of the inhibitor. In addition, we have found from double-mutant cycle analysis on T58AE60A and T58DE60A that there are interaction energies between residues 58 and 60 in the free inhibitor of 0.5 +/- 0.2 and 0.4 +/- 0.1 kcal mol(-1) (measured for T58AE60A and T58DE60A, respectively) that increase substantially in the complex with subtilisin BPN' to 2.1 +/- 0.1 and 4.2 +/- 0.2 kcal mol(-1), respectively. The hydrogen bonding and salt bridge network within the active site loop, which is a characteristic of potato inhibitor I family of inhibitors, is essential for the inhibitory activity of the protein.