Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.
Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.
复制标题
L-亮氨酸膦酸抑制蛋白水解气单胞菌的氨肽酶。
DOI：
10.1021/bi0100891
复制
发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Petsko,G
中科院分区：
文献类型：
--
作者：
Stamper,C;Bennett,B;Edwards,T;Holz,RC;Ringe,D;Petsko,G
研究了过渡态类似物抑制剂亮氨酸膦酸（LPA）与水解蛋白单胞菌（AAP）亮氨酸氨基肽酶的相互作用性质。结果表明，LPA在pH为8.0、aki为6.6 μM时是一种竞争性抑制剂。加入LPA后[CoCo(AAP)]、[CoZn(AAP)]和[ZnCo(AAP)]在pH 7.5下的电子吸收光谱表明，LPA在双核活性位点与这两种金属离子相互作用。对AAP的Co（II）取代形式的EPR研究表明，在加入LPA后，[CoZn(AAP)]和[ZnCo(AAP)]中Co（II）离子的环境变得高度不对称和受限，并清楚地表明LPA与这两种金属离子相互作用。在2.1 Å分辨率下测定了AAP与LPA配合的x射线晶体结构。x射线晶体学数据表明，LPA与AAP双核活性位点的两个金属中心相互作用，没有单氧原子桥。因此，LPA以η-1,2-μ-膦酸盐的形式与AAP的双核活性位点结合，其中一个配体与n端胺提供的第二个金属离子结合。对含磷酸盐过渡态类似物与单金属和双金属肽酶结合的结构比较，有助于深入了解桥接双金属肽酶对第二金属离子的需求。根据本文给出的光谱学和x射线晶体学数据以及已有报道的AAP催化机理数据，提出了AAP催化水解反应的新催化机理。
The nature of the interaction of the transition-state analogue inhibitorl-leucinephosphonic acid (LPA) with the leucine aminopeptidase fromAeromonas proteolytica(AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with aKiof 6.6 μM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 Å resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an η-1,2-μ-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.