Kojima S, Hisano Y, Miura K
Institute for Biomolecular Science, Gakushuin University, Tokyo, Mejiro, 171-8588, Japan.
Biochem Biophys Res Commun. 2001 Mar;281(5):1271-6. doi: 10.1006/bbrc.2001.4515.
Pleurotus ostreatus proteinase A inhibitor 1 (POIA1), which is composed of 76 residues without disulfide bridges, is a unique inhibitor in that it exhibits sequence similarity to the propeptides of subtilisins. In order to elucidate the inhibitory mechanism of POIA1, we constructed an expression system for a synthetic POIA1 gene. The wild-type POIA1 was found to inhibit subtilisin BPN' with an inhibitor constant (K(i)) of 3.2 x 10(-9) M, but exhibited a time-dependent decrease of inhibitory activity as a consequence of degradation by the protease, showing that the wild-type POIA1 was a temporary inhibitor when subtilisin BPN' was used as a target protease. Since POIA1 shows sequence similarity to the propeptide of subtilisin, which is known to inhibit the protease via its C-terminal region, the C-terminal six residues of POIA1 were replaced with those of the propeptide of subtilisin BPN'. The mutated POIA1 inhibited subtilisin BPN' with a K(i) value of 2.8 x 10(-11) M and did not exhibit time-dependent decrease of inhibitory activity, showing about 100-fold increases in binding affinity for, and resistance to, the protease. These results clearly indicate that the C-terminal region of POIA1 plays an important role in determining the inhibitory activity toward the protease, and that the increase in binding ability to the protease is closely related to resistance to proteolytic degradation. Therefore, the inhibitory properties of POIA1 can be altered by mutation of its C-terminal region.
糙皮侧耳蛋白酶A抑制剂1（POIA1）由76个残基组成，无二硫键，是一种独特的抑制剂，因为它与枯草杆菌蛋白酶的前肽具有序列相似性。为了阐明POIA1的抑制机制，我们构建了一个合成POIA1基因的表达系统。发现野生型POIA1抑制枯草杆菌蛋白酶BPN'，其抑制常数（K(i)）为3.2×10(-9) M，但由于被蛋白酶降解，其抑制活性呈现出时间依赖性下降，这表明当以枯草杆菌蛋白酶BPN'作为靶蛋白酶时，野生型POIA1是一种临时抑制剂。由于POIA1与枯草杆菌蛋白酶的前肽具有序列相似性，已知该前肽通过其C末端区域抑制蛋白酶，因此将POIA1的C末端六个残基替换为枯草杆菌蛋白酶BPN'前肽的残基。突变后的POIA1抑制枯草杆菌蛋白酶BPN'的K(i)值为2.8×10(-11) M，并且没有呈现出抑制活性的时间依赖性下降，表明其对蛋白酶的结合亲和力和抗性增加了约100倍。这些结果清楚地表明，POIA1的C末端区域在决定对蛋白酶的抑制活性中起重要作用，并且与蛋白酶的结合能力的增加与对蛋白水解降解的抗性密切相关。因此，POIA1的抑制特性可以通过其C末端区域的突变而改变。
