Hohl Mathias, Stintzi Annick, Schaller Andreas
From the Institute of Plant Physiology and Biotechnology, University of Hohenheim, D-70593 Stuttgart, Germany.
From the Institute of Plant Physiology and Biotechnology, University of Hohenheim, D-70593 Stuttgart, Germany
J Biol Chem. 2017 Apr 14;292(15):6389-6401. doi: 10.1074/jbc.M117.775445. Epub 2017 Feb 21.
The propeptides of subtilisin-like serine proteinases (subtilases, SBTs) serve dual functions as intramolecular chaperones that are required for enzyme folding and as inhibitors of the mature proteases. SBT propeptides are homologous to the I9 family of protease inhibitors that have only been described in fungi. Here we report the identification and characterization of subtilisin propeptide-like inhibitor 1 (SPI-1) from Sequence similarity and the shared β-α-β-β-α-β core structure identified SPI-1 as a member of the I9 inhibitor family and as the first independent I9 inhibitor in higher eukaryotes. SPI-1 was characterized as a high-affinity, tight-binding inhibitor of subtilase SBT4.13 with and values in the picomolar range. SPI-1 acted as a stable inhibitor of SBT4.13 over the physiologically relevant range of pH, and its inhibitory profile included many other SBTs from plants but not bovine chymotrypsin or bacterial subtilisin A. Upon binding to SBT4.13, the C-terminal extension of SPI-1 was proteolytically cleaved. The last four amino acids at the newly formed C terminus of SPI-1 matched both the cleavage specificity of SBT4.13 and the consensus sequence of SBTs at the junction of the propeptide with the catalytic domain. The data suggest that the C terminus of SPI-1 acts as a competitive inhibitor of target proteases as it remains bound to the active site in a product-like manner. SPI-1 thus resembles SBT propeptides with respect to its mode of protease inhibition. However, in contrast to SBT propeptides, SPI-1 could not substitute as a folding assistant for SBT4.13.
枯草杆菌蛋白酶样丝氨酸蛋白酶（枯草杆菌蛋白酶，SBTs）的前肽具有双重功能，既是酶折叠所需的分子内伴侣，又是成熟蛋白酶的抑制剂。SBT前肽与仅在真菌中描述过的I9蛋白酶抑制剂家族同源。在此，我们报告了从[具体来源未给出]中鉴定和表征枯草杆菌蛋白酶前肽样抑制剂1（SPI-1）的过程。序列相似性以及共享的β-α-β-β-α-β核心结构确定SPI-1为I9抑制剂家族的成员，并且是高等真核生物中的首个独立I9抑制剂。SPI-1被表征为枯草杆菌蛋白酶SBT4.13的高亲和力、紧密结合抑制剂，其解离常数（KD）和抑制常数（KI）值在皮摩尔范围内。在生理相关的pH范围内，SPI-1作为SBT4.13的稳定抑制剂起作用，其抑制谱包括许多来自植物的其他SBTs，但不包括牛胰凝乳蛋白酶或细菌枯草杆菌蛋白酶A。与SBT4.13结合后，SPI-1的C末端延伸被蛋白水解切割。SPI-1新形成的C末端的最后四个氨基酸既符合SBT4.13的切割特异性，也符合前肽与催化结构域交界处SBTs的共有序列。数据表明，SPI-1的C末端作为靶蛋白酶的竞争性抑制剂起作用，因为它以产物样方式保持与活性位点结合。因此，SPI-1在蛋白酶抑制模式方面类似于SBT前肽。然而，与SBT前肽不同，SPI-1不能替代作为SBT4.13的折叠辅助剂。
