Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies

Abstract

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met 11-NH 2. The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln 6-Phe 7, Phe 7-Phe 8, and Gly 9-Leu 10 and neurotensin (NT) at Pro 10-Tyr 11 and Tyr 11-Ile 12. Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe 8-Gly 9 and Gly 9-Leu 10 to release C-terminal tri- and dipeptide (ratio=4:1). The hydrolysis was Cl − dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr 11-Ile 12 to release Ile 12-Leu 13. Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu 5-enkephalin. The K i values in μM were as follows: for ACE, bradykinin=0.4, angiotensin I=4, SP=25, SP free acid=2, NT=14, and Met 5-enkephalin=450, and for NEP, bradykinin=162, angiotensin I=36, SP=190, NT=39, and Met 5−enkephalin=22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.