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首页> 外文期刊>Biochemistry >SUBSTRATE BINDING AND CATALYSIS BY UBIQUITIN C-TERMINAL HYDROLASES - IDENTIFICATION OF TWO ACTIVE SITE RESIDUES
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SUBSTRATE BINDING AND CATALYSIS BY UBIQUITIN C-TERMINAL HYDROLASES - IDENTIFICATION OF TWO ACTIVE SITE RESIDUES

机译：泛素C末端水合物对基质的结合和催化作用-两种活性残基的鉴定

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摘要

Ubiquitin C-terminal hydrolases (UCH's) are a newly-defined class of thiol proteases implicated in the proteolytic processing of polymeric ubiquitin, They are important for the generation of monomeric ubiquitin, the active component of the eukaryotic ubiquitin-dependent proteolytic system, There are at least three mammalian isozymes which are tissue specific and developmentally regulated. To study the structure and functional roles of these highly homologous enzymes, we have subcloned and overexpressed two of these isozymes, UCH-L1 and UCH-L3. Here, we report their purification, physical characteristics, and the mutagenesis of UCH-L1. Site-directed mutagenesis of UCH-L1 reveals that C90 and H161 are involved in catalytic rate enhancement. Data from circular dichroic and Raman spectroscopy, as well as secondary structure prediction algorithms, indicate that both isozymes have a significant amount of alpha-helix (>35%), and contain no disulfide bonds. Both enzymes are reasonably stable, undergoing a reversible thermal denaturation at 52 degrees C. These transitions are characterized by thermodynamic parameters typical of single domain globular proteins. Substrate binding affinity to UCH-L3 was directly measured by equilibrium gel filtration (K-d = 0.5 mu M), and the results are similar to the kinetically determined K-m for ubiquitin ethyl ester (0.6 mu M). The binding is primarily electrostatic in nature and indicates the existence of a specific and extensive binding site for ubiquitin on the surface of the enzyme.
机译：泛素C末端水解酶（UCH's）是一类新定义的硫醇蛋白酶，与聚合泛素的蛋白水解过程有关，它们对于产生单体泛素（真核泛素依赖性蛋白水解系统的活性成分）很重要。至少三种组织特异性和发育调节的哺乳动物同工酶。为了研究这些高度同源的酶的结构和功能作用，我们已经亚克隆并过表达了其中两种同功酶UCH-L1和UCH-L3。在这里，我们报告其纯化，物理特征，和诱变的UCH L1。 UCH-L1的定点诱变表明C90和H161参与催化速率的提高。来自圆二色性和拉曼光谱以及二级结构预测算法的数据表明，两种同工酶均具有大量的α-螺旋（> 35％），并且不包含二硫键。两种酶都相当稳定，在52摄氏度时经历可逆的热变性。这些转变的特征是单域球状蛋白的典型热力学参数。通过平衡凝胶过滤（K-d = 0.5μM）直接测量底物对UCH-L3的结合亲和力，其结果类似于动力学测定的泛素乙酯K-m（0.6μM）。结合本质上主要是静电的，表明泛素在酶表面上存在特异性和广泛的结合位点。

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