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PMID:1320019

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Citation

Wei, L, Clauser, E, Alhenc-Gelas, F and Corvol, P (1992) The two homologous domains of human angiotensin I-converting enzyme interact differently with competitive inhibitors. J. Biol. Chem. 267:13398-405

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.

Links

PubMed

Keywords

Angiotensin I/metabolism; Angiotensin-Converting Enzyme Inhibitors/metabolism; Angiotensin-Converting Enzyme Inhibitors/pharmacology; Animals; Binding Sites; CHO Cells; Captopril/metabolism; Captopril/pharmacology; Chlorides; Cricetinae; Enalapril/analogs & derivatives; Enalapril/metabolism; Enalapril/pharmacology; Enalaprilat/metabolism; Enalaprilat/pharmacology; Humans; Indoles/metabolism; Indoles/pharmacology; Kinetics; Lisinopril; Mutation; Peptidyl-Dipeptidase A/genetics; Peptidyl-Dipeptidase A/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:ACE

enables

GO:0008144: drug binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACE

enables

GO:0008241: peptidyl-dipeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:ACE

enables

GO:0008237: metallopeptidase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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