Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family
Essential roles of zinc ligation and enzyme dimerization for catalysis in the aminoacylase-1/M20 family
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DOI：
10.1074/jbc.m304233200
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发表时间：
2003-11-07
影响因子：
4.8
通讯作者：
Ménard, R
中科院分区：
文献类型：
--
作者：
Lindner, HA;Lunin, VV;Ménard, R
氨基酰基酶-1(Acy1)/M20家族的氨基酰基酶和外肽酶以单体或同源二聚体的形式存在。它们包含一个锌结合结构域和第二个结构域，在后一种情况下介导二聚作用。通过X射线结晶学和定点突变研究了这两个结构域在人Acy1(HAcy1)催化中的作用。本文报道的hAcy1突变体中的双核锌中心与相关酶中的双锌中心的结构比较表明，Acy1/M20家族中的锌连接存在差异。突变分析支持锌离子、邻近的谷氨酸和二聚化结构域中的组氨酸的催化作用。通过补充hAcy1的不同活性位点突变体，我们表明催化发生在二聚体界面。对单体同系物--肽酶V的结构的重新解释表明，结构域插入模仿了二聚化。我们得出结论，单体和二聚体Acy1/M20家族成员共享一个涉及这两个酶结构域的独特的活性位点结构。本研究可能为改进同源羧基肽酶G2在抗体导向酶前药治疗中的应用提供手段。
Members of the aminoacylase-1 (Acy1)/M20 family of aminoacylases and exopeptidases exist as either monomers or homodimers. They contain a zinc-binding domain and a second domain mediating dimerization in the latter case. The roles that both domains play in catalysis have been investigated for human Acy1 (hAcy1) by x-ray crystallography and by site-directed mutagenesis. Structure comparison of the dinuclear zinc center in a mutant of hAcy1 reported here with dizinc centers in related enzymes points to a difference in zinc ligation in the Acy1/M20 family. Mutational analysis supports catalytic roles of zinc ions, a vicinal glutamate, and a histidine from the dimerization domain. By complementing different active site mutants of hAcy1, we show that catalysis occurs at the dimer interface. Reinterpretation of the structure of a monomeric homolog, peptidase V, reveals that a domain insertion mimics dimerization. We conclude that monomeric and dimeric Acy1/M20 family members share a unique active site architecture involving both enzyme domains. The study may provide means to improve homologous carboxypeptidase G2 toward application in antibody-directed enzyme prodrug therapy.