Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism.
In the RAS (renin-angiotensin system), Ang I (angiotensin I) is cleaved by ACE (angiotensin-converting enzyme) to form Ang II (angiotensin II), which has effects on blood pressure, fluid and electrolyte homoeostasis. We have examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, the homologue of ACE, ACE2, and NEP (neprilysin). The activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates. Ang I was effectively cleaved by NEP to Ang (1-7) (kcat/K(m) of 6.2x10(5) M(-1) x s(-1)), but was a poor substrate for ACE2 (kcat/K(m) of 3.3x10(4) M(-1) x s(-1)). Ang (1-9) was a better substrate for NEP than ACE (kcat/K(m) of 3.7x10(5) M(-1) x s(-1) compared with kcat/K(m) of 6.8x10(4) M(-1) x s(-1)). Ang II was cleaved efficiently by ACE2 to Ang (1-7) (kcat/K(m) of 2.2x10(6) M(-1) x s(-1)) and was cleaved by NEP (kcat/K(m) of 2.2x10(5) M(-1) x s(-1)) to several degradation products. In contrast with a previous report, Ang (1-7), like Ang I and Ang (1-9), was cleaved with a similar efficiency by both the N- and C-domains of ACE (kcat/K(m) of 3.6x10(5) M(-1) x s(-1) compared with kcat/K(m) of 3.3x10(5) M(-1) x s(-1)). The two active sites of ACE exhibited negative co-operativity when either Ang I or Ang (1-7) was the substrate. In addition, a range of ACE inhibitors failed to inhibit ACE2. These kinetic data highlight that the flux of peptides through the RAS is complex, with the levels of ACE, ACE2 and NEP dictating whether vasoconstriction or vasodilation will predominate.
Rice
Gillian I
GI
Proteolysis Research Group, School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, UK.
Thomas
Daniel A
DA
Grant
Peter J
PJ
Turner
Anthony J
AJ
Hooper
Nigel M
NM
D000803:Angiotensin I / Q000378:metabolism; D000804:Angiotensin II / Q000378:metabolism; D000085962:Angiotensin-Converting Enzyme 2; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000809:Angiotensins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D015260:Neprilysin / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism*; D013379:Substrate Specificity
D000806:Angiotensin-Converting Enzyme Inhibitors; D000809:Angiotensins; D010446:Peptide Fragments; D000804:Angiotensin II; D000803:Angiotensin I; D002268:Carboxypeptidases; D007703:Peptidyl-Dipeptidase A; C000705307:ACE2 protein, human; D000085962:Angiotensin-Converting Enzyme 2; D015260:Neprilysin; C118790:angiotensin I (1-7)