Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts
Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts
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DOI：
10.1021/bi971264
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发表时间：
1997-10-14
期刊：
影响因子：
2.9
通讯作者：
Menard, R
中科院分区：
文献类型：
--
作者：
Nagler, DK;Storer, AC;Menard, R
将组织蛋白酶B与木瓜蛋白酶家族的其它半胱氨酸蛋白酶区分开的主要特征是存在大的插入环，称为闭合环，其占据酶的S'亚位点。该环主要通过与酶的其余部分的两个接触保持在适当位置，包括环上的残基His 110和Arg 116，它们分别与Asp 22和Asp 224形成盐桥。使用定点诱变和内部淬灭荧光（IQF）底物研究了该环对组织蛋白酶B内肽酶活性的影响。与组织蛋白酶L和木瓜蛋白酶相比，野生型组织蛋白酶B对底物Abz-AFRSAAQ-EDDnp和Abz-QVVAGA-EDDnp显示出差的活性。对于含有单突变D22 A、H110 A、R116 A和D224 A的组织蛋白酶B，观察到k（cat）/K-M明显增加。然而，对于其中两个环与酶的接触都被破坏的突变体观察到最高活性。对于三突变体D22 A/H110 A/R116 A，Abz-AFRSAAQ-EDDnp的水解获得了12 × 10（5）M-1 s（-1）的最佳k（cat）/K-M值，其对应于相对于野生型组织蛋白酶B增加600倍，并且接近用组织蛋白酶L或木瓜蛋白酶观察到的活性水平。通过比较，突变对Cbz-FR-MCA的水解几乎没有影响。突变对活性的pH依赖性的影响还表明，通常观察到的组织蛋白酶B的pH活性曲线的复杂性与封闭环的存在有关。内肽酶活性的主要增加归因于环的“灵活性”的增加，并表明闭塞环可能会移动时，内肽酶底物结合到酶，这些相互作用在调节内肽酶活性的可能贡献和组织蛋白酶B活性在生理或病理条件下的影响进行了讨论。
The main feature distinguishing cathepsin B from other cysteine proteases of the papain family is the presence of a large insertion loop, termed the occluding loop, which occupies the S' subsites of the enzyme. The loop is held in place mainly by two contacts with the rest of the enzyme, involving residues His110 and Arg116 on the loop that form salt bridges with Asp22 and Asp224, respectively. The influence of this loop on the endopeptidase activity of cathepsin B has been investigated using site-directed mutagenesis and internally quenched fluorogenic (IQF) substrates. Wild-type cathepsin B displays poor activity against the substrates Abz-AFRSAAQ-EDDnp and Abz-QVVAGA-EDDnp as compared to cathepsin L and papain. Appreciable increases in k(cat)/K-M were observed for cathepsin B containing the single mutations D22A, H110A, R116A, and D224A. The highest activity however is observed for mutants where both loop to enzyme contacts are disrupted. For the triple-mutant D22A/H110A/R116A, an optimum k(cat)/K-M value of 12 x 10(5) M-1 s(-1) was obtained for hydrolysis of Abz-AFRSAAQ-EDDnp, which corresponds to a 600-fold increase relative to wild-type cathepsin B and approaches the level of activity observed with cathepsin L or papain. By comparison, the mutations have little effect on the hydrolysis of Cbz-FR-MCA. The influence of the mutations on the pH dependency of activity also indicates that the complexity of pH activity profiles normally observed for cathepsin B is related to the presence of the occluding loop. The major increase in endopeptidase activity is attributed to an increase in loop ''flexibility'' and suggests that the occluding loop might move when an endopeptidase substrate binds to the enzyme, The possible contribution of these interactions in regulating endopeptidase activity and the implications for cathepsin B activity in physiological or pathological conditions are discussed.