Subsite specificity of memapsin 2 (β-secretase):: Implications for inhibitor design
Subsite specificity of memapsin 2 (β-secretase):: Implications for inhibitor design
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DOI：
10.1021/bi015546s
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发表时间：
2001-08-28
期刊：
影响因子：
2.9
通讯作者：
Tang, J
中科院分区：
文献类型：
--
作者：
Turner, RT;Koelsch, G;Tang, J
膜蛋白酶2是称为β-分泌酶的蛋白酶，其对β-淀粉样前体蛋白的作用导致β-淀粉样（AP）肽的产生。由于A β在大脑中的积累是阿尔茨海默病发病机制中的关键事件，因此memapsin 2是设计抑制性药物的重要靶标。在这里，我们描述的残基偏好的memapsin 2的亚位点。通过MALDI-TOF质谱法定量的底物混合物的相对初始裂解速率确定8个亚位点中每个亚位点中残基的相对k（cat）/K-M值。我们发现每个亚位点可以容纳多个残基。S-1亚位点是最严格的，优选的残基顺序为Leu > Phe > Met > Tyr。其它亚位点的选择性如下：S-2，Asp > Asn > Met; S-3，Ile >瓦尔> Leu; S-4，Glu > Gln > Asp; S-1 '，Met > Glu > Gln > Ala; S-2'，瓦尔> Ile > Ala; S-3 '，Leu > Trp > Ala; S-4'，Asp > Glu > Trp。一般来说，S亚位点比S'亚位点更特异。发现包含八个最有利的残基（Glu-Ile-Asp-Leu-Met-Val-Leu-Asp）的肽被水解，具有迄今为止对于memapsin 2观察到的最高k（cat）/K-M值。在四个亚位点的残基偏好也进行了研究结合memapsin 2的组合抑制剂库。在10种紧密结合抑制剂中，共有偏好如下：S2，Asp和Glu; S3，Leu和Ile; S-2 '，瓦尔;和S-3'，Glu和Gln。从共有残基设计的抑制剂OM 00 -3，Glu-Leu-Asp-Leu*Ala-Val-Glu-Phe（其中星号代表羟丙甲酰基转移状态等排体）被发现是迄今为止报道的最有效的memapsin 2抑制剂（Ki为3.1 × 10（-10）M）。OM 00 -3与memapsin 2结合的分子模型揭示了抑制剂侧链与酶之间的相互作用相对于先前的抑制剂OM 99 -2的关键改进。
Memapsin 2 is the protease known as beta -secretase whose action on beta -amyloid precursor protein leads to the production of the P-amyloid (AP) peptide. Since the accumulation of A beta in the brain is a key event in the pathogenesis of Alzheimer's disease, memapsin 2 is an important target for the design of inhibitory drugs. Here we describe the residue preference for the subsites of memapsin 2. The relative k(cat)/K-M values of residues in each of the eight subsites were determined by the relative initial cleavage rates of substrate mixtures as quantified by MALDI-TOF mass spectrometry. We found that each subsite can accommodate multiple residues. The S-1 subsite is the most stringent, preferring residues in the order of Leu > Phe > Met > Tyr. The preferences of other subsites are the following: S2, Asp > Asn > Met; S-3, Ile > Val > Leu; S-4, Glu > Gln > Asp; S-1', Met > Glu > Gln > Ala; S-2', Val > Ile > Ala; S-3', Leu > Trp > Ala; S-4', Asp > Glu > Trp. In general, S subsites are more specific than the S' subsites. A peptide comprising the eight most favored residues (Glu-Ile-Asp-Leu-Met-Val-Leu-Asp) was found to be hydrolyzed with the highest k(cat)/K-M value so far observed for memapsin 2. Residue preferences at four subsites were also studied by binding of memapsin 2 to a combinatorial inhibitor library. From 10 tight binding inhibitors, the consensus preferences were as follows: S2, Asp and Glu; S3, Leu and Ile; S-2', Val; and S-3', Glu and Gln. An inhibitor, OM00-3, Glu-Leu-Asp-Leu*Ala-Val-Glu-Phe (where the asterisk represents the hydroxyethylene tansition-state isostere), designed from the consensus residues, was found to be the most potent inhibitor of memapsin 2 so far reported (K-i of 3.1 x 10(-10) M). A molecular model of OM00-3 binding to memapsin 2 revealed critical improvement of the interactions between inhibitor side chains with enzyme over a previous inhibitor, OM99-2.