N-domain selectivity of angiotensin I-converting enzyme as assessed by structure-function studies of its highly selective substrate, N-acetyl-seryl-aspartyl-lysyl-proline.
The physiological functions of angiotensin I-converting enzyme (ACE) are not limited to its cardiovascular role. ACE constantly degrades N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a natural circulating regulator of the hematopoietic stem cell proliferation, and thereby may be involved in hematopoietic stem cell regulation. AcSDKP is hydrolyzed 50-fold faster by the N-domain active site compared to the C-domain active site. The aim of the present study was to investigate which aminoacid residues from AcSDKP are required to ensure N-domain specificity. Several peptides were designed by progressively increasing the length of the peptidic chain from a tripeptide to a pentapeptide. Kinetic studies of the wild-type ACE and of the two ACE mutants containing a single active domain (N- or C-domain) were performed using Bz (benzoyl) Asp-Lys-Pro, benzoyl-glycyl (Bz-Gly)-Asp-Lys-Pro, and Bz-Gly-Ser-Asp-Lys-Pro (with its intermediate product Bz-Gly-Ser-Asp) as substrates. The unexpected importance of an aspartic acid in the P1 position was discovered, as well as the interaction of the P2 and P3 positions in the substrate to increase or decrease N-domain specificity. Substrates longer than five residues may involve interdependence between subsites. Finally, the discovery of highly specific and novel N-domain substrates cannot be predicted from single subsite mapping, but may require other approaches such as combinatorial peptide libraries.
Institut National de la Sante et de la Recherche Medicale, Unite 36, College de France, Paris.
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Michaud
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Chauvet
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Corvol
D001665:Binding Sites; D007700:Kinetics; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D009842:Oligopeptides; D007703:Peptidyl-Dipeptidase A; C058504:goralatide