Inhibitory potency and specificity of subtilase-like pro-protein convertase (SPC) prodomains
Inhibitory potency and specificity of subtilase-like pro-protein convertase (SPC) prodomains
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DOI：
10.1074/jbc.m107467200
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发表时间：
2002-03-08
影响因子：
4.8
通讯作者：
Day, R
中科院分区：
文献类型：
--
作者：
Fugère, M;Limperis, PC;Day, R
SPC（枯草杆菌蛋白酶样前蛋白转化酶）是负责组成性和调节性分泌途径的许多前体蛋白的蛋白水解加工的酶家族。SPC本身作为非活性酶原合成。SPC的活化通过前结构域的分子内自催化去除发生。SPC前结构域已被提议作为模板用于开发有效且特异的SPC抑制剂。在这项研究中，我们研究了特异性和效力的完整的前结构域和短的C-末端前结构域肽的每个SPC高度纯化，可溶性酶制剂的人SPC 1，SPC 6和SPC 7。前结构域肽和完整前结构域的进展曲线动力学分析显示在低纳摩尔范围内的竞争性抑制曲线。完整的前结构域比C-末端前结构域肽有效5-100倍，表明N-末端决定簇参与识别过程。然而，完整的前结构域和前结构域肽仅对其同源酶表现出部分特异性。Ala-扫描结构活性研究表明，在P-4，P-5和P-6位置的碱性残基的抑制SPC 1的重要性。相反，P-6和P-7中的疏水残基以及P-6和P-7中的碱性残基对于SPC 7的抑制是关键的。我们的数据表明，使用前结构域作为反式作用的特异性抑制剂将是有限的有用性，除非修改成更具体的化合物。
The SPCs (subtilisin-like pro-protein convertases) are a family of enzymes responsible for the proteolytic processing of numerous precursor proteins of the constitutive and regulated secretory pathways. SPCs are themselves synthesized as inactive zymogens. Activation of SPCs occurs via the intramolecular autocatalytic removal of the prodomain. SPC prodomains have been proposed as templates in the development of potent and specific SPC inhibitors. In this study, we investigated the specificity and potency of complete prodomains and short C-terminal prodomain peptides of each SPC on highly purified, soluble enzyme preparations of human SPC1, SPC6, and SPC7. Progress curve kinetic analysis of prodomain peptides and complete prodomains showed competitive inhibitory profiles in the low nano-molar range. Complete prodomains were 5-100 times more potent than C-terminal prodomain peptides, suggesting that N-terminal determinants are involved in the recognition process. However, complete prodomains and prodomain peptides exhibit only a partial specificity toward their cognate enzyme. Ala-scan structure activity studies indicated the importance of basic residues in the P-4, P-5, and P-6 positions for inhibition of SPC1. In contrast, hydrophobic residues in P-6 and P-7, as well as basic residues in P-6 and P-7, were critical for inhibition of SPC7. Our data demonstrated that the use of prodomains as specific inhibitors acting in trans would be of limited usefulness, unless modified into more specific compounds.