The new alpha-amino acid N-omega-hydroxy-nor-L-arginine: A high-affinity inhibitor of arginase well adapted to bind to its manganese cluster
The new alpha-amino acid N-omega-hydroxy-nor-L-arginine: A high-affinity inhibitor of arginase well adapted to bind to its manganese cluster
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DOI：
10.1021/ja970285o
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发表时间：
1997-04-30
影响因子：
15
通讯作者：
Zimmermann, JL
中科院分区：
文献类型：
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作者：
Custot, J;Moali, C;Zimmermann, JL
哺乳动物精氨酸酶特别需要Mn（II）-Mn（II）簇用于其催化活性，将L-精氨酸（L-arg）水解为L-鸟氨酸和尿素。[1]最近，大鼠肝脱氢酶的晶体结构表明，两个Mn离子由来自蛋白质的两个羧酸酯侧链和一个水分子（或氢氧根离子）桥接。2有人提出，L-arg与Mn簇合物紧密结合，反应的过渡态是一种四面体物质，由金属桥连氢氧化物对L-arg的胍碳进行亲核攻击而产生（图1）。2，3目前关于Mn簇对不同于L-arg的分子的可接近性的数据非常少，并且硼酸盐是迄今为止描述的唯一显著改变酶的EPR谱的化合物。4抑制剂通常是探索酶活性位点的有趣工具。长期以来，最为人所知的抑制剂是L-缬氨酸，其Ki值适中（在毫摩尔范围内）。5最近，Nω-羟基-L-精氨酸（NOHA）6（方案1），一种从L-精氨酸生物合成NO的中间体，和一些Nω-羟基-L-R-氨基酸7已被证明是更有效的抑制剂，Ki值在20-50 µM范围内。已经提出了一个模型的相互作用，这些抑制剂与辅酶A酶，这假设，他们可能是过渡态类似物类型，N-OH基团能够取代的辅酶A酶锰簇的羟基配体。6a，7在此模型的基础上，我们发现Nω-羟基-去甲-L-精氨酸（nor-NOHA）应该是一种非常好的辅酶A酶抑制剂，非常适合通过其N-OH功能与Mn簇相互作用本文报道了（i）Nω-羟基-去甲-L-精氨酸及其同系物Nω-羟基-高-L-精氨酸的首次合成，（ii）NOHA、homo-NOHA和nor-NOHA对脱氢酶的抑制作用的比较，和（iii）这些化合物与纯化的大鼠肝脱氢酶的相互作用的EPR研究。他们表明，nor-NOHA不仅是最好的抑制剂（Ki <0.5 µM），而且还特异性地改变了酶的EPR谱。Nω-羟基-高-L-精氨酸（高-NOHA）（方案1）9按照先前描述的NOHA合成方法由L-赖氨酸合成。8 Nor-NOHA 9是由方案1所示的反应中的L-谷氨酰胺获得的（15%总产率）。关键步骤是用次溴酸钠使NR-Boc-L-谷氨酰胺脱羧，生成中间体NR-Boc-nor-L-鸟氨酸，其易于被保护为Nδ-
Mammalian arginases specifically require a Mn (II)-Mn (II) cluster for their catalytic activity, the hydrolysis of L-arginine (L-arg) to L-ornithine and urea. 1 Quite recently, the crystal structure of rat liver arginase has shown that the two Mn ions are bridged by two carboxylate side chains of aspartates from the protein and a water molecule (or hydroxide ion). 2 It has been proposed that L-arg binds in close proximity of the Mncluster and that the transition state of the reaction is a tetrahedral species resulting from nucleophilic attack of the metal-bridging hydroxide at the guanidinium carbon of L-arg (Figure 1). 2, 3 Very few data are presently available about the accessibility of the Mn-cluster to molecules different from L-arg, and borate was the only compound described so far to significantly modify the EPR spectrum of arginase. 4 Inhibitors often are interesting tools to explore enzyme active sites. For arginase, for a long time the best known inhibitor was L-valine, which exhibits a modest Ki value (in the millimolar range). 5 More recently, Nω-hydroxy-L-arginine (NOHA) 6 (Scheme 1), an intermediate in the biosynthesis of NO from L-arginine, and some Nω-hydroxy-L-R-aminoacids7 have been shown to act as much more potent inhibitors of arginases with Ki values in the 20-50 µM range. A model has been proposed for the interaction of those inhibitors with arginase, which postulated that they could be of the transition state analog type, the N-OH group being able to replace the hydroxo ligand of the arginase Mn-cluster. 6a, 7 On the basis of this model, it appeared to us that Nω-hydroxy-nor-L-arginine (nor-NOHA) should be a very good arginase inhibitor, well suited to interact with the Mn cluster Via its N-OH function (better than NOHA, Figure 1).This paper reports preliminary results about (i) the first synthesis of Nω-hydroxy-nor-L-arginine and its homolog Nω-hydroxy-homo-L-arginine,(ii) a comparison of the inhibitory effects of NOHA, homo-NOHA, and nor-NOHA toward arginases, and (iii) EPR studies of the interaction of these compounds with purified rat liver arginase. They show that nor-NOHA not only is the best inhibitor (Ki) 0.5 µM) but also specifically modifies the arginase EPR spectrum. Nω-hydroxy-homo-L-arginine (homo-NOHA)(Scheme 1) 9 was synthesized from L-lysine following a procedure previously described for the synthesis of NOHA. 8 Nor-NOHA9 was obtained from L-glutamine from reactions shown in Scheme 1 (15% overall yield). The key step was the decarboxylation of NR-Boc-L-glutamine by sodium hypobromite to intermediate NR-Boc-nor-L-ornithine which was readily protected as a Nδ-