Sivaraman J, Nägler D K, Zhang R, Ménard R, Cygler M
Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, Québec, H4P 2R2, Canada.
J Mol Biol. 2000 Jan 28;295(4):939-51. doi: 10.1006/jmbi.1999.3410.
Human cathepsin X is one of many proteins discovered in recent years through the mining of sequence databases. Its sequence shows clear homology to cysteine proteases from the papain family, containing the characteristic residue patterns, including the active site. However, the proregion of cathepsin X is only 38 residues long, the shortest among papain-like enzymes, and the cathepsin X sequence has an atypical insertion in the regions proximal to the active site. This protein was recently expressed and partially characterized biochemically. Unlike most other cysteine proteases from the papain family, procathepsin X is incapable of autoprocessing in vitro but can be processed under reducing conditions by exogenous cathepsin L. Atypically, the mature enzyme is primarily a carboxypeptidase and has extremely poor endopeptidase activity. We have determined the three-dimensional structure of the procathepsin X at 1.7 A resolution. The overall structure of the mature enzyme is characteristic for enzymes of the papain superfamily, but contains several novel features. Most interestingly, the short proregion binds to the enzyme with the aid of a covalent bond between the cysteine residue in the proregion (Cys10p) and the active site cysteine residue (Cys31). This is the first example of a zymogen in which the inhibition of enzyme's proteolytic activity by the proregion is achieved through a reversible covalent modification of the active site nucleophile. Such mode of binding requires less contact area between the proregion and the enzyme than observed in other procathepsins, and no auxiliary binding site on the enzyme surface is used. A three-residue insertion in a highly conserved region, just prior to the active site cysteine residue, confers a significantly different shape on the S' subsites, compared to other proteases from papain family. The 3D structure provides an explanation for the rather unusual carboxypeptidase activity of this enzyme and confirms the predictions based on homology modeling. Another long insertion in the cathepsin X amino acid sequence forms a beta-hairpin pointing away from the active site. This insertion, thought to be an equivalent of cathepsin B occluding loop, is located on the side of the protein, distant from the substrate binding site.
人组织蛋白酶X是近年来通过序列数据库挖掘发现的众多蛋白质之一。其序列与木瓜蛋白酶家族的半胱氨酸蛋白酶具有明显的同源性，包含特征性的残基模式，包括活性位点。然而，组织蛋白酶X的前肽区仅38个残基长，是类木瓜蛋白酶中最短的，并且组织蛋白酶X序列在活性位点近端区域有一个非典型插入。该蛋白最近已被表达并进行了部分生化特性分析。与木瓜蛋白酶家族的大多数其他半胱氨酸蛋白酶不同，组织蛋白酶原X在体外不能自动加工，但在还原条件下可被外源性组织蛋白酶L加工。不同寻常的是，成熟酶主要是一种羧肽酶，其内切肽酶活性极差。我们已经确定了组织蛋白酶原X在1.7埃分辨率下的三维结构。成熟酶的整体结构是木瓜蛋白酶超家族酶的特征，但包含几个新特征。最有趣的是，短前肽区借助前肽区中的半胱氨酸残基（Cys10p）与活性位点半胱氨酸残基（Cys31）之间的共价键与酶结合。这是一个酶原的首个例子，其中前肽区通过活性位点亲核试剂的可逆共价修饰来抑制酶的蛋白水解活性。这种结合模式比在其他组织蛋白酶原中观察到的前肽区与酶之间需要更少的接触面积，并且未使用酶表面的辅助结合位点。在活性位点半胱氨酸残基之前的高度保守区域中的一个三残基插入，与木瓜蛋白酶家族的其他蛋白酶相比，赋予S'亚位点显著不同的形状。三维结构为该酶相当不寻常的羧肽酶活性提供了解释，并证实了基于同源建模的预测。组织蛋白酶X氨基酸序列中的另一个长插入形成了一个远离活性位点的β-发夹结构。这个插入被认为等同于组织蛋白酶B的封闭环，位于蛋白质的一侧，远离底物结合位点。
