Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250
Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250
复制标题
DOI：
10.1107/s090744490901779x
复制
发表时间：
2009-08-01
期刊：
影响因子：
--
通讯作者：
Yoshimoto, Tadashi
中科院分区：
文献类型：
--
作者：
Fournie-Zaluski, Marie-Claude;Poras, Herve;Yoshimoto, Tadashi
从大肠杆菌中纯化的氨基肽酶 N (APN; EC 3.4.11.2) 已用光学纯氨基膦抑制剂 PL250 H(3)N(+)-CH(CH(3))-P(O)(OH)-CH(2)-CH(CH(2)Ph)-CONH-CH(CH(2)Ph)CO(2)(-) 结晶，模拟了过渡态水解反应。 PL250 抑制 APN 的 K(i) 为 1.5-2.2 nM，其与大肠杆菌 APN 复合的三维结构显示其与催化位点的 S(1)、S(1)' 和 S(2)' 亚位点相互作用。在该结构中，Zn离子与APN的His297、His301和Glu320以及PL250的次膦酸部分的两个O原子进行五配位。这些 O 原子之一还参与与 Tyr381 的氢键，支持了该氨基酸在稳定酶促过程过渡态中的作用。与二肽衍生抑制剂贝他汀相比，次膦酸锌结合的强度和 S(2)' 亚位点的占据使 PL250 的亲和力增加了 100 倍 (K(i) = 4.1 x 10(-6) M）。因此，PL250 C 末端苯丙氨酸的去除导致亲和力大幅下降（K(i) = 2.17 x 10(-7) M）。此外，观察到抑制剂的 C 末端羧基与 APN 活性位点的氨基酸没有直接相互作用。有趣的是，PL250 对大肠杆菌 APN 和哺乳动物酶表现出相同的抑制效力，这表明该复合物的结构可以用作合理设计各种人类 APN 抑制剂的模板，以研究这种氨肽酶在各种病理中的作用。
Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H(3)N(+)-CH(CH(3))-P(O)(OH)-CH(2)-CH(CH(2)Ph)-CONH-CH(CH(2)Ph)CO(2)(-), which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a K(i) of 1.5-2.2 nM and its three-dimensional structure in complex with E. coli APN showed its interaction with the S(1), S(1)' and S(2)' subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S(2)' subsite account for the 100-fold increase in affinity of PL250 compared with the dipeptide-derived inhibitor bestatin (K(i) = 4.1 x 10(-6) M). Accordingly, the removal of the C-terminal phenylalanine of PL250 resulted in a large decrease in affinity (K(i) = 2.17 x 10(-7) M). Furthermore, it was observed that the C-terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies.