Deddish P A, Wang L X, Jackman H L, Michel B, Wang J, Skidgel R A, Erdös E G
Department of Pharmacology, University of Illinois College of Medicine, Chicago, USA.
J Pharmacol Exp Ther. 1996 Dec;279(3):1582-9.
Somatic angiotensin I converting enzyme (ACE; kininase II) has two active sites, in two (N and C) domains. We studied the active centers with separate N-domain ACE (N-ACE), testicular C-domain ACE (germinal ACE) and, as control, renal somatic ACE. Germinal ACE cleaved the nonapeptide bradykinin about two times faster than N-ACE in 20 mM Cl-. Bradykinin1-7 was hydrolyzed further to bradykinin1-5 by N-ACE four times faster in the absence of Cl-, but at 300 mM Cl- the C-domain hydrolyzed it twice as fast. The hematopoietic system regulatory peptide acetyl-Ser-Asp-Lys-Pro was split to two dipeptides by N-ACE, depending on the chloride concentration, 8 to 24 times faster than by germinal ACE; at 100 mM Cl-, the Kcat with N-ACE was eight times higher. One millimolar 1-fluoro-2,4-dinitrobenzene inhibited germinal ACE 96% but it inhibited N-ACE by only 31%. [3H]Ramiprilat was displaced by other unlabeled ACE inhibitors to establish their relative affinities. Captopril had the lowest IC50 (0.5 nM) with N-ACE and the highest IC50 (8.3 nM) with the germinal ACE. The IC50 values of ramiprilat and quinaprilat were about the same with both active sites. The association and dissociation constants of [3H]ramiprilat indicated faster association with and faster dissociation from N-ACE than from germinal ACE. After exposure to alkali or moderate heat, somatic ACE was cleaved by plasmin and kallikrein, releasing N-ACE and apparently inactivating the C-domain. These studies affirm the differences in the activity, stability and inhibition of the two active sites of ACE.
体细胞血管紧张素I转换酶（ACE；激肽酶II）在两个（N和C）结构域中有两个活性位点。我们用分离的N结构域ACE（N-ACE）、睾丸C结构域ACE（生殖ACE）以及作为对照的肾体细胞ACE研究了活性中心。在20 mM Cl-中，生殖ACE切割九肽缓激肽的速度比N-ACE快约两倍。在无Cl-的情况下，N-ACE将缓激肽1-7进一步水解为缓激肽1-5的速度快四倍，但在300 mM Cl-时，C结构域水解它的速度快两倍。造血系统调节肽乙酰-Ser-Asp-Lys-Pro被N-ACE切割成两个二肽，这取决于氯化物浓度，比生殖ACE快8至24倍；在100 mM Cl-时，N-ACE的催化常数Kcat高八倍。1 mM 1-氟-2,4-二硝基苯抑制生殖ACE 96%，但仅抑制N-ACE 31%。用其他未标记的ACE抑制剂取代[3H]雷米普利拉以确定它们的相对亲和力。卡托普利对N-ACE的IC50最低（0.5 nM），对生殖ACE的IC50最高（8.3 nM）。雷米普利拉和喹那普利拉的IC50值在两个活性位点大致相同。[3H]雷米普利拉的结合和解离常数表明，与生殖ACE相比，它与N-ACE的结合更快，解离也更快。暴露于碱或适度加热后，体细胞ACE被纤溶酶和激肽释放酶切割，释放出N-ACE，显然使C结构域失活。这些研究证实了ACE两个活性位点在活性、稳定性和抑制方面的差异。
