Substrate-related potent inhibitors of brain metalloendopeptidase.
Substrate-related potent inhibitors of brain metalloendopeptidase.
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脑金属内肽酶的底物相关有效抑制剂。
DOI：
10.1021/bi00402a015
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发表时间：
1988
期刊：
影响因子：
2.9
通讯作者：
Molineaux,CJ
中科院分区：
文献类型：
--
作者：
Orlowski,M;Michaud,C;Molineaux,CJ
1987年9月17日摘要：大鼠脑金属内肽酶（EC 3.4。24.15）从几种较大的阿片肽产生亮氨酸和蛋氨酸脑啡肽，并能够降解许多神经肽。合成了底物相关的7V-（1-羧基-3-苯基丙基）肽衍生物，并测试了其酶抑制作用。这些衍生物中最好的是N-[1（RS ′）-羧基-3-苯基丙基]-Ala-Ala-Tyr-对-氨基苯甲酸酯，以竞争性方式抑制酶，Ki为16 nM。数据表明，JV-（1-羧基-3-苯基丙基）部分的羧基与活性中心锌原子配位，并且抑制剂的其余部分对于与酶的底物识别位点的相互作用是必需的。用羧甲基取代1-羧基-3-苯丙基使抑制效力降低了3个数量级以上，强调了疏水苯基对抑制剂在St亚位点与疏水口袋结合的重要性。用Ala残基取代Tyr残基使抑制效力降低20倍以上。与Sj'亚位点相互作用的残基结构的改变可引起抑制作用的60倍以上的变化。抑制剂对膜结合金属内肽酶（“脑啡肽酶”，EC 3.4）无效或仅具有弱抑制作用。24.11），一种在兔肾中高度活跃的酶，但也存在于脑中。数据表明，在酶中存在延伸的结合位点，其中残基与S1、S1和S3'亚位点相互作用，这在很大程度上决定了抑制剂结合。这些发现与从其与合成和天然肽的相互作用的研究中推断的酶的特异性一致[Orlowski，M.，Michaud，C.，& Chu，TG（1983）Eur. J. Biochem.135，81-88]。在该实验室中的先前工作已经导致从大鼠脑中鉴定和纯化主要与脑匀浆的可溶性蛋白质部分相关的金属内肽酶（Orlowski等人，1983年）。最近还鉴定了与脑膜部分（包括纯化的突触体膜）相关的酶的膜结合形式（阿克等人，1986年）。被称为内肽酶24.15的酶与嗜热菌蛋白酶样膜结合金属内肽酶（EC 3.4）明显不同。24.11，内肽酶24.11; Kerr & Kenny，1974; Orlowski & Wilk，1981）。不同于已知与“脑啡肽酶”相同的内肽酶24.11（Almenoff等人，1981; Fulcher等人，1982），一种广泛分布于动物组织中的酶，内肽酶24.15在脑、垂体和睾丸中具有高活性，而在其它外周组织如肝、肾、肺或脾中几乎没有活性。此外，与主要被认为具有降解功能，特别是在脑脑啡肽（“脑啡肽酶”）的降解中的内肽酶24.11不同，内肽酶24.15由于其特异性而潜在地参与一些生物活性肽的加工和形成以及其他生物活性肽的降解。因此，酶的可溶性和膜结合形式都有效地将几种肽（包括强啡肽1 ™ 8、α-和β-新内啡肽以及Met-enk-Arg-Gly-Leu 1）分别转化为生物活性五肽Leu-enk和Met-enk（Chu & Orlowski，1985;阿克（Acker）等人，1986年）。该酶还显示出对其他几种生物活性肽的高亲和力，如缓激肽、神经降压素和血管紧张素I，将这些肽转化为
Revised Manuscript Received September 17, 1987 abstract: Rat brain metalloendopeptidase (EC 3.4. 24.15) generates Leu-and Met-enkephalinfrom several larger opioid peptides and is capable of degrading a number of neuropeptides. Substrate-related 7V-(1-carboxy-3-phenylpropyl) peptide derivatives were synthesized and tested for enzyme inhibition. The best of these derivatives, N-[1 (RS')-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-/>-aminobenzoate, inhibited the enzyme in a competitive manner with a K, of 16 nM. The data indicate that the carboxyl group of the JV-(1-carboxy-3-phenylpropyl) moiety coordinates with the activesite zinc atom and that the remaining part of the inhibitor is necessary for interaction with the substrate recognition site of the enzyme. Replacement of the 1-carboxy-3-phenylpropyl group by a carboxymethyl group decreased the inhibitory potency by more than 3 orders of magnitude, emphasizing the importance of the hydrophobic phenyl group for inhibitor binding to a hydrophobic pocketat the St subsite. Replacement of the Tyr residue by an Ala residue decreased the inhibitory potency by more than 20-fold. Changes in the structure of the residue interactingwith the Sj'subsite could cause a more than 60-fold change in inhibition. The inhibitors were either ineffective or only weakly inhibitory against membrane-bound metalloendopeptidase (“enkephalinase”, EC 3.4. 24.11), an enzyme highly active in rabbit kidney but also presentin brain. The data indicate the presence of an extended binding site in the enzyme with residues interacting with S,, S/, and S3'subsites largely determining inhibitor binding. These findings are consistent with the specificity of the enzyme deduced from studies of its interaction with synthetic and natural peptides [Orlowski, M., Michaud, C., & Chu, TG (1983) Eur. J. Biochem. 135, 81-88].^ Revious work inthis laboratory has led tothe identification and purification from rat brain of a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates (Orlowski et al., 1983). A membrane-bound form of the enzyme, associated with brainmembrane fractions, including purified synaptosomal membranes has also been recently identified (Acker et al., 1986). The enzyme to be referred to as endopeptidase 24.15, is distinctly different from thermolysin-like membrane-boundmetalloendopeptidase (EC 3.4. 24.11, endopeptidase 24.11; Kerr & Kenny, 1974; Orlowski & Wilk, 1981) with respect to bothspecificity and molecular properties. Unlike endopeptidase 24.11, known to be identical with “enkephalinase”(Almenoff et al., 1981; Fulcher et al., 1982), an enzyme widely distributed in animal tissues, endopeptidase 24.15 is highly active in brain, pituitary, and testis with little activity in other peripheral tissues, such as liver, kidney, lung, or spleen. Also, unlike endopeptidase 24.11 which is primarily considered to have a degradative function, especially in the degradation of brain enkephalins (“enkephalinase”), endopeptidase 24.15, due to its specificity, is potentially involved in processing and formation of some bioactive peptides and degradation of others. Thus, both the soluble and membrane-bound forms of the enzyme efficiently converted several peptides, including dynorphin1™ 8, a- and jS-neoendorphin, and Met-enk-Arg-Gly-Leu1 into the bioactive pentapeptides Leu-enk and Met-enk, respectively (Chu & Orlowski, 1985; Acker et al., 1986). The enzyme also showed a high affinity toward several other bioactive peptides, such as bradykinin, neurotensin, and angiotensin I, converting these