Kojima S, Minagawa T, Miura K
Institute for Biomolecular Science, Gakushuin University, Mejiro, Tokyo, Japan.
FEBS Lett. 1997 Jul 7;411(1):128-32. doi: 10.1016/s0014-5793(97)00678-9.
The propeptide of subtilisin-family proteases is known to exhibit inhibitory activity toward a cognate protease in addition to its function as an intramolecular chaperone. For detailed investigation of its inhibitory properties, the propeptide of subtilisin BPN' was produced in Escherichia coli. Inhibitory activity measurements and electrophoresis showed that the propeptide was a temporary inhibitor, which was initially potent but was gradually degraded by subtilisin BPN' through specific intermediates. The main cleavage site was identified as Glu53-Lys54, with minor sites at Thr17-Met18 and Met21-Ser22, which were located in turn regions of the propeptide in the complex with subtilisin BPN'. Since the isolated propeptide has been shown not to form a tertiary structure, these results indicate that main digestions proceed through proteolytic attack of subtilisin toward the accessible sites of the propeptide in the complex with subtilisin. Therefore, replacement of Glu53 at the main cleavage site by Asp, which is a less favorable amino acid than Glu for subtilisin, makes the propeptide a more resistant temporary inhibitor.
已知枯草杆菌蛋白酶家族蛋白酶的前肽除了作为分子内伴侣发挥功能外，还对同源蛋白酶表现出抑制活性。为了详细研究其抑制特性，枯草杆菌蛋白酶BPN'的前肽在大肠杆菌中产生。抑制活性测量和电泳表明，该前肽是一种临时抑制剂，最初具有很强的活性，但会被枯草杆菌蛋白酶BPN'通过特定中间体逐渐降解。主要切割位点被确定为Glu53-Lys54，次要位点位于Thr17-Met18和Met21-Ser22，它们依次位于与枯草杆菌蛋白酶BPN'形成复合物的前肽的转角区域。由于已证明分离出的前肽不会形成三级结构，这些结果表明主要的消化过程是通过枯草杆菌蛋白酶对与枯草杆菌蛋白酶形成复合物的前肽的可及位点进行蛋白水解攻击来进行的。因此，将主要切割位点的Glu53替换为Asp（对于枯草杆菌蛋白酶来说，Asp是比Glu更不利的氨基酸），可使前肽成为更具抗性的临时抑制剂。
