Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection.
Hydrogen bond residue positioning in the 599-611 loop of thimet oligopeptidase is required for substrate selection.
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DOI：
10.1111/j.1742-4658.2008.06685.x
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发表时间：
2008-11
期刊：
影响因子：
--
通讯作者：
Wolfson AJ
中科院分区：
文献类型：
--
作者：
Bruce LA;Sigman JA;Randall D;Rodriguez S;Song MM;Dai Y;Elmore DE;Pabon A;Glucksman MJ;Wolfson AJ
Thiet寡肽酶（EC 3.4.24.15; TOP）是涉及许多生理肽的加工的Zn（II）内肽酶。虽然其在选择和加工肽中的作用尚未完全了解，但据信衬于底物结合位点的柔性环区域允许酶符合不同结构的底物。本研究描述了TOP的突变形式，其中环区599-611中的Gly或Tyr残基被单独或组合取代，以阐明TOP的底物选择机制。在Tyr 605和Tyr 612突变后观察到的kcat降低表明这些残基有助于大多数底物的有效切割。建模研究表明，铰链弯曲运动将使Tyr 612和Tyr 605在裂解的肽键的H键距离内支持这一作用。因此，分子模拟研究支持的关键作用，这种酶的过渡态稳定的Tyr 605。有趣的是，动力学参数表明，缓激肽衍生物的加工明显不同于其他基板测试表明，可采用替代催化机制，为这个特定的基板。数据表明，Tyr 605和Tyr 612对于该底物的水解都不是必需的。相对于其他底物，缓激肽衍生物也不受环中Gly突变的影响。这种区别表明，甘氨酸残基在环中的作用是适当地定向这些Tyr残基，以适应不同的底物结构。这也开启了某些底物可以被酶的开放形式切割的可能性。
Thimet oligopeptidase (EC 3.4.24.15; TOP) is a Zn(II) endopeptidase implicated in the processing of numerous physiological peptides. Though its role in selecting and processing peptides is not fully understood, it is believed that flexible loop regions lining the substrate-binding site allow the enzyme to conform to substrates of varying structure. The present study describes mutant forms of TOP in which Gly or Tyr residues in loop region 599-611 were replaced, individually and in combination, to elucidate the mechanism of substrate selection by TOP. Decreases in kcat observed upon mutation of Tyr605 and Tyr612 demonstrate that these residues contribute to the efficient cleavage of most substrates. Modeling studies showing that a hinge-bend movement would bring both Tyr612 and Tyr605 within H-bond distance of the cleaved peptide bond supports this role. Thus, molecular modeling studies support a key role in transition-state stabilization of this enzyme by Tyr605. Interestingly, kinetic parameters showed that a bradykinin derivative is processed distinctly from the other substrates tested suggesting an alternative catalytic mechanism may be employed for this particular substrate. The data demonstrate that neither Tyr605 nor Tyr612 are necessary for the hydrolysis of this substrate. Relative to other substrates, the bradykinin derivative is also unaffected by Gly mutations in the loop. This distinction suggests that the role of glycine residues in the loop is to properly orient these Tyr residues in order to accommodate varying substrate structures. This also opens the possibility that certain substrates may be cleaved by an open form of the enzyme.
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影响因子：
4.8
作者：
Cummins, PM;Pabon, A;Glucksman, MJ
通讯作者：
Glucksman, MJ
影响因子：
8
作者：
HOLLAND, DR;HAUSRATH, AC;MATTHEWS, BW
通讯作者：
MATTHEWS, BW
DOI：
10.1006/bbrc.1999.0251
发表时间：
1999-02-24
影响因子：
3.1
作者：
Portaro, FCV;Gomes, MD;de Camargo, ACM
通讯作者：
de Camargo, ACM
影响因子：
3.5
作者：
Oliveira, V;Araújo, MC;Ferro, ES
通讯作者：
Ferro, ES
影响因子：
2.9
作者：
PIEROTTI, A;DONG, KW;ROBERTS, JL
通讯作者：
ROBERTS, JL