Structural Basis for Substrate Recognition and Hydrolysis by Mouse Carnosinase CN2*
Structural Basis for Substrate Recognition and Hydrolysis by Mouse Carnosinase CN2*
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DOI：
10.1074/jbc.m801657200
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发表时间：
2008-10
影响因子：
4.8
通讯作者：
H. Unno;T. Yamashita;S. Ujita;Nobuaki Okumura;H. Otani;A. Okumura;K. Nagai;M. Kusunoki
中科院分区：
文献类型：
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作者：
H. Unno;T. Yamashita;S. Ujita;Nobuaki Okumura;H. Otani;A. Okumura;K. Nagai;M. Kusunoki
L-肌氨酸是一种生物活性二肽(β-丙氨酰-L-组氨酸)，存在于哺乳动物组织中，包括中枢神经系统，具有潜在的神经保护和神经递质功能。到目前为止，在哺乳动物中已经克隆了两种L肌肽水解酶(CN1和CN2)，它们被归类为M20家族的金属肽酶。CN2的酶活性需要Mn2+，并且CN2被一种不可水解的底物类似物Bestatin抑制。本文报道了Bestatin与锌离子形成1.7Å的络合物和与Mn2+形成2.3Å的络合物的晶体结构。CN2是非晶体不对称单元中的同源二聚体，Mn2+和Zn2+配合物在整体结构上非常相似。每个亚基由两个结构域组成：结构域A与Bestatin和两个金属离子络合，结构域B为二聚体的形成提供主要界面。与A结构域结合的Bestatin分子与另一个亚基的B结构域的几个残基相互作用，这些相互作用可能是酶活性所必需的。由于Bestatin分子不能与主体水结合，底物结合需要结构域A和B之间的构象灵活性。活性部位结构和底物结合模型为CN2及其相关酶的酶活性和底物专一性提供了结构基础。
l-Carnosine is a bioactive dipeptide (β-alanyl-l-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of l-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn2+, and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn2+ at a resolution of 1.7Å and that with Mn2+ at 2.3Å. CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn2+ and Zn2+ complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.