Cunha Carlos Eduardo L, Magliarelli Helena de Fátima, Paschoalin Thaysa, Nchinda Aloysius T, Lima Jackson C, Juliano Maria A, Paiva Paulo B, Sturrock Edward D, Travassos Luiz R, Carmona Adriana K
Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil.
Biol Chem. 2009 Sep;390(9):931-40. doi: 10.1515/BC.2009.105.
Dipeptidyl carboxypeptidase from Escherichia coli (EcDcp) is a zinc metallopeptidase with catalytic properties closely resembling those of angiotensin I-converting enzyme (ACE). However, EcDcp and ACE are classified in different enzyme families (M3 and M2, respectively) due to differences in their primary sequences. We cloned and expressed EcDcp and studied in detail the enzyme's S(3) to S(1)' substrate specificity using positional-scanning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides. These peptides contain ortho-aminobenzoic acid (Abz) and 2,4-dinitrophenyl (Dnp) as donor/acceptor pair. In addition, using FRET substrates developed for ACE [Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH] as well as natural ACE substrates (angiotensin I, bradykinin, and Ac-SDKP-OH), we show that EcDcp has catalytic properties very similar to human testis ACE. EcDcp inhibition studies were performed with the ACE inhibitors captopril (K(i)=3 nM) and lisinopril (K(i)=4.4 microM) and with two C-domain-selective ACE inhibitors, 5-S-5-benzamido-4-oxo-6-phenylhexanoyl-L-tryptophan (kAW; K(i)=22.0 microM) and lisinopril-Trp (K(i)=0.8 nM). Molecular modeling was used to provide the basis for the differences found in the inhibitors potency. The phylogenetic relationship of EcDcp and related enzymes belonging to the M3 and M2 families was also investigated and the results corroborate the distinct origins of EcDcp and ACE.
来自大肠杆菌的二肽基羧肽酶（EcDcp）是一种锌金属肽酶，其催化特性与血管紧张素I转换酶（ACE）极为相似。然而，由于其一级序列的差异，EcDcp和ACE被归类于不同的酶家族（分别为M3和M2）。我们克隆并表达了EcDcp，并使用荧光共振能量转移（FRET）肽的位置扫描合成组合（PS-SC）文库，详细研究了该酶对S(3)至S(1)'底物的特异性。这些肽含有邻氨基苯甲酸（Abz）和2,4-二硝基苯基（Dnp）作为供体/受体对。此外，我们使用为ACE开发的FRET底物[Abz-FRK(Dnp)P-OH、Abz-SDK(Dnp)P-OH和Abz-LFK(Dnp)-OH]以及天然ACE底物（血管紧张素I、缓激肽和Ac-SDKP-OH），证明EcDcp具有与人类睾丸ACE非常相似的催化特性。我们使用ACE抑制剂卡托普利（K(i)=3 nM）和赖诺普利（K(i)=4.4 microM）以及两种C结构域选择性ACE抑制剂5-S-5-苯甲酰胺基-4-氧代-6-苯基己酰基-L-色氨酸（kAW；K(i)=22.0 microM）和赖诺普利-色氨酸（K(i)=0.8 nM）进行了EcDcp抑制研究。分子建模为抑制剂效力差异提供了依据。我们还研究了EcDcp与属于M3和M2家族的相关酶的系统发育关系，结果证实了EcDcp和ACE的不同起源。
