Combined use of selective inhibitors and fluorogenic substrates to study the specificity of somatic wild-type angiotensin-converting enzyme.
Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. Studies on the selectivity of these ACE domains towards either substrates or inhibitors have mostly relied on the use of mutants or isolated domains of ACE. To determine directly the selectivity properties of each ACE domain, working with wild-type enzyme, we developed an approach based on the combined use of N-domain-selective and C-domain-selective ACE inhibitors and fluorogenic substrates. With this approach, marked differences in substrate selectivity were revealed between rat, mouse and human somatic ACE. In particular, the fluorogenic substrate Mca-Ala-Ser-Asp-Lys-DpaOH was shown to be a strict N-domain-selective substrate of mouse ACE, whereas with rat ACE it displayed marked C-domain selectivity. Similar differences in selectivity between these ACE species were also observed with a new fluorogenic substrate of ACE, Mca-Arg-Pro-Pro-Gly-Phe-Ser-Pro-DpaOH. In support of these results, changes in amino-acid composition in the binding site of these three ACE species were pinpointed. Together these data demonstrate that the substrate selectivity of the N-domain and C-domain depends on the ACE species. These results raise concerns about the interpretation of functional studies performed in animals using N-domain and C-domain substrate selectivity data derived only from human ACE.
Jullien
Nicolas D
ND
CEA, Département d'Ingénerie et d'Etudes des Protéines, Gif/Yvette, France.
Cuniasse
Philippe
P
Georgiadis
Dimitris
D
Yiotakis
Athanasios
A
Dive
Vincent
V
D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D004338:Drug Combinations; D005456:Fluorescent Dyes; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009842:Oligopeptides / Q000494:pharmacology*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000378:metabolism; D010721:Phosphinic Acids / Q000494:pharmacology*; D051381:Rats; D011917:Rats, Inbred Lew; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000806:Angiotensin-Converting Enzyme Inhibitors; D004338:Drug Combinations; D005456:Fluorescent Dyes; D009842:Oligopeptides; D010721:Phosphinic Acids; C118733:RXP 407; C476531:RXPA380; D007703:Peptidyl-Dipeptidase A