Lenarcic B, Krishnan G, Borukhovich R, Ruck B, Turk V, Moczydlowski E
Departments of Biochemistry and Molecular Biology, J. Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.
J Biol Chem. 2000 May 19;275(20):15572-7. doi: 10.1074/jbc.M001406200.
The type 1 domain of thyroglobulin is a protein module (Thyr-1) that occurs in a variety of secreted and membrane proteins. Several examples of Thyr-1 modules have been previously identified as inhibitors of the papain family of cysteine proteinases. Saxiphilin is a neurotoxin-binding protein from bullfrog and a homolog of transferrin with a pair of such Thyr-1 modules located in the N-lobe. Saxiphilin is now characterized as a potent inhibitor of three cysteine proteinases as follows: papain, human cathepsin B, and cathepsin L. The stoichiometry of enzyme inhibition reveals that both Thyr-1 domains of saxiphilin inhibit papain (apparent K(i) = 1. 72 nm), but only one of these domains inhibits cathepsin B (K(i) = 1. 67 nm) and cathepsin L (K(i) = 0.02 nm). Physical association of saxiphilin and papain blocked from turnover at the active-site cysteine residue can be detected by cross-linking with glutaraldehyde. The rate of association of saxiphilin and cathepsin B is strongly pH-dependent with an optimum at pH 5.2, reflecting control by at least two H(+)-titratable groups. These results further demonstrate that various Thyr-1 domains are selective inhibitors of cysteine proteinases with utility in the study of protein interactions and degradation.
甲状腺球蛋白的1型结构域是一种蛋白质模块（Thyr-1），存在于多种分泌蛋白和膜蛋白中。先前已鉴定出几个Thyr-1模块的例子是半胱氨酸蛋白酶木瓜蛋白酶家族的抑制剂。嗜石蛋白是一种来自牛蛙的神经毒素结合蛋白，是转铁蛋白的同源物，在N叶中有一对这样的Thyr-1模块。嗜石蛋白现在被表征为三种半胱氨酸蛋白酶的有效抑制剂，如下所示：木瓜蛋白酶、人组织蛋白酶B和组织蛋白酶L。酶抑制的化学计量表明，嗜石蛋白的两个Thyr-1结构域都抑制木瓜蛋白酶（表观K(i)=1.72nm），但这些结构域中只有一个抑制组织蛋白酶B（K(i)=1.67nm）和组织蛋白酶L（K(i)=0.02nm）。通过与戊二醛交联可以检测到嗜石蛋白和在活性位点半胱氨酸残基处被阻止周转的木瓜蛋白酶的物理结合。嗜石蛋白与组织蛋白酶B的结合速率强烈依赖于pH值，在pH5.2时达到最佳，这反映了至少两个可滴定H(+)基团的控制。这些结果进一步证明，各种Thyr-1结构域是半胱氨酸蛋白酶的选择性抑制剂，在蛋白质相互作用和降解研究中具有实用性。
