Metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase
Metal ion substitution in the catalytic site greatly affects the binding of sulfhydryl-containing compounds to leucyl aminopeptidase
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DOI：
10.1021/bi052069v
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发表时间：
2006-03-14
期刊：
影响因子：
2.9
通讯作者：
Mura, U
中科院分区：
文献类型：
--
作者：
Cappiello, M;Alterio, V;Mura, U
牛透镜亮氨酰氨肽酶（bIdehyde）是一种同六聚体金属肽酶，其N端的疏水氨基酸是其主要的底物，每个亚基含有两个Zn ~（2+）离子。它们可以被具有不同交换动力学的其他二价阳离子取代。蛋白质易交换位点（位点1）可被Zn 2+、Mn 2+、Mg 2+或Co 2+占据，而紧密结合位点（位点2）可被Zn 2+或Co 2+占据。我们最近报道，将Mn 2+引入位点1产生了针对CysGly的新的生物活性[Cappiello，M.，等人（2004）Biochem.J.378，35-44]，相反，其不被（Zn/Zn）酶水解。这一发现，虽然揭示了谷胱甘肽代谢中的一个潜在的特定作用，提出了一个问题的分子所需的功能是酶的底物。为了阐明酶与含巯基衍生物的相互作用，制备了（Zn/Zn）-和（Mn/Zn）双金属形式，并进行了功能-结构研究。因此，进行了两种酶形式的各种化合物的动力学分析;确定了与拟肽衍生物佐芬普利拉复合的（Zn/Zn）bIl的晶体结构，并对CysGly-（Zn/Zn）bIl复合物进行了建模研究。这种组合的方法提供了深入了解的相互作用的bIbis与含巯基的衍生物，显示在网站I的金属交换调节结合这些分子，可能会导致酶底物或抑制剂，这取决于金属的性质。
Bovine lens leucyl aminopeptidase (bILAP), a homohexameric metallopeptidase preferring bulky and hydrophobic amino acids at the N-terminus of (di)peptides, contains two Zn2+ ions per subunit that are essential for catalytic activity. They may be replaced by other divalent cations with different exchange kinetics. The protein readily exchangeable site (site 1) can be occupied by Zn2+, Mn2+, Mg2+, or Co2+, while the tight binding site (site 2) can be occupied by Zn2+ or Co2+. We recently reported that introduction of Mn2+ into site 1 generates a novel activity of bILAP toward CysGly [Cappiello, M., et al. (2004) Biochem. J. 378, 35-44], which in contrast is not hydrolyzed by the (Zn/Zn) enzyme. This finding, while disclosing a potential specific role for blLAP in glutathione metabolism, raised a question about the features required for molecules to be a substrate for the enzyme. To clarify the interaction of the enzyme with sulfhydryl-containing derivatives, (Zn/Zn)- and (Mn/Zn)bILAP forms were prepared and functional-structural studies were undertaken. Thus, a kinetic analysis of various compounds with both enzyme forms was performed; the crystal structure of (Zn/Zn)bILAP in complex with the peptidomimetic derivative Zofenoprilat was determined, and a modeling study on the CysGly-(Zn/Zn)bILAP complex was carried out. This combined approach provided insight into the interaction of bILAP with sulfhydryl-containing derivatives, showing that the metal exchange in site I modulates binding to these molecules that may result in enzyme substrates or inhibitors, depending on the nature of the metal.