The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors.

Abstract

The endothelial angiotensin I-converting enzyme (ACE; EC 3.4.15.1) has recently been shown to contain two large homologous domains (called here the N and C domains), each being a zinc-dependent dipeptidyl carboxypeptidase. To further characterize the two active sites of ACE, we have investigated their interaction with four competitive ACE inhibitors, which are all potent antihypertensive drugs. The binding of [3H] trandolaprilat to the two active sites was examined using the wild-type ACE and four ACE mutants each containing only one intact domain, the other domain being either deleted or inactivated by point mutation of the zinc-coordinating histidines. In contrast with all the previous studies, which suggested the presence of a single high affinity inhibitor binding site in ACE, the present study shows that both the N and C domains of ACE contain a high affinity inhibitor binding site (KD = 3 and 1 X 10(-10) M, respectively, at pH 7.5, 4 degrees C, and 100 mM NaCl). Chloride stabilizes the enzyme-inhibitor complex for each domain primarily by slowing its dissociation rate, as the k-1 values of the N and C domains are markedly decreased (about 30- and 1100-fold, respectively) by 300 mM NaCl. At high chloride concentrations, the chloride effect is much greater for the C domain than for the N domain resulting in a higher affinity of this inhibitor for the C domain. In addition, the inhibitory potency of captopril (C), enalaprilat (E), and lisinopril (L) for each domain was assayed by hydrolysis of Hip-His-Leu. Their Ki values for the two domains are all within the nanomolar range, indicating that they are all highly potent inhibitors for both domains. However, their relative potencies are different for the C domain (L greater than E greater than C) and the N domain (C greater than E greater than L). The different inhibitor binding properties of the two domains observed in the present study provide strong evidence for the presence of structural differences between the two active sites of ACE.