Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15 - Identification of active site histidine and glutamate residues
Zinc coordination and substrate catalysis within the neuropeptide processing enzyme endopeptidase EC 3.4.24.15 - Identification of active site histidine and glutamate residues
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DOI：
10.1074/jbc.274.23.16003
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发表时间：
1999-06-04
影响因子：
4.8
通讯作者：
Glucksman, MJ
中科院分区：
文献类型：
--
作者：
Cummins, PM;Pabon, A;Glucksman, MJ
内肽酶EC 5.4.24.15(EP24.15)是一种锌金属内肽酶，广泛分布于脑、垂体和性腺中。其底物特异性包括许多重要的生理活性神经肽，如神经降压素、缓激肽和促性腺激素释放激素，促性腺激素释放激素是生殖的主要调节肽。在研究EP24.15的结构和功能时，我们采用了。体外突变和随后的蛋白质表达为了从基因上剖析该酶并使我们能够深入了解底物结合和催化的机制，将EP24.15的序列与以前通过X射线结晶学解决并用作哺乳动物金属内肽酶模型的细菌同源物进行比较，表明了保守的残基。EP24.15的活性部位显示出一个HEXXH基序，这是锌金属酶的一个共同特征，突变证实了该基序中残基(His(473)、Glu(474)和His(477))对结合和催化的重要性，第三种可能的金属配体被认为与His(473)和His(477)直接配位到活性部位的锌离子，被确定为Glu(502)。这些残基的保守改变极大地降低了酶对假定的生理底物和合成猝灭的荧光底物的活性，以及特定的活性部位定向抑制剂N-[1-(RS)-羧基-3-苯丙基]-丙氨酸-丙氨酸-酪氨酸-对氨基苯甲酸酯的结合，我们已经证明其结合依赖于活性部位锌离子的存在和可能的配位。这些研究有助于更全面地了解EP24.15的催化机制，并有助于合理设计这类酶的抑制剂和药理药物。
Endopeptidase EC 5.4.24.15 (EP24.15) is a zinc metalloendopeptidase that is broadly distributed within the brain, pituitary, and gonads. Its substrate specificity includes a number of physiologically important neuropeptides such as neurotensin, bradykinin, and gonadotropin-releasing hormone, the principal regulatory peptide for reproduction. In studying the structure and function of EP24.15, we have employed in. vitro mutagenesis and subsequent protein expression to genetically dissect the enzyme and allow us to glean insight into the mechanism of substrate binding and catalysis, Comparison of the sequence of EP24.15 with bacterial homologues previously solved by x-ray crystallography and used as models for mammalian metalloendopeptidases, indicates conserved residues. The active site of EP24.15 exhibits an HEXXH motif, a common feature of zinc metalloenzymes, Mutations have confirmed the importance, for binding and catalysis, of the residues (His(473), Glu(474), and His(477)) within this motif, A third putative metal ligand, presumed to coordinate directly to the active site zinc ion in concert with His(473) and His(477), has been identified as Glu(502). Conservative alterations to these residues drastically reduces enzymatic activity against both a putative physiological substrate and a synthetic quenched fluorescent substrate as well as binding of the specific active site-directed inhibitor, N-[1-(RS) -carboxy-3-phenylpropyl] -Ala-Ala-Tyr-p-aminobenzoate, the binding of which we have shown to be dependent upon the presence, and possibly coordination, of the active site zinc ion. These studies contribute to a more complete understanding of the catalytic mechanism of EP24.15 and will aid in rational design of inhibitors and pharmacological agents for this class of enzymes.