Hydrolysis of substance P and neurotensin by converting enzyme and neutral endopeptidase

Abstract

Angiotensin I converting enzyme (ACE) and neutral endopeptidase (“enkephalinase”; NEP), were 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. NEP hydrolyzed 0.1 mM SP, NT and their C-terminal fragments at the following rates (μmol/min/mg): SP 1–11=7.8, SP 4–11=11.7, SP 5–11=15.4, SP 6–11=15.6, SP 8–11=6.7, NT 1–13=2.9, and NT 8–13=4.0. Purified ACE 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 mainly C-terminal tripeptide from SP methyl ester, but only dipeptide from SP free acid. Modification of arginine residues in ACE with cyclohexanedione or butanedione similarly inhibited hydrolysis of SP, bradykinin and Bz-Gly-Phe-Arg (80–93%) indicating an active site arginine is required for hydrolysis of SP. ACE hydrolyzed NT at Tyr 11-Ile 12 to release Ile 12-Leu 13. SP, NT and their derivatives (0.1 mM) were cleaved by ACE at the following rates (μmol/min/mg): SP 1–11=1.2, SP methyl ester=0.7, SP free acid=8.5, SP 4–11=2.4, SP 5–11=0.9, SP 6–11=1.4, SP 8–11=0, NT 1–13=0.2, and NT 8–13=1.3. Peptide substrates were used as inhibitors of ACE (substrate=FA-Phe-Gly-Gly) and NEP (substrate=Leu 5-enkephalin). K i values (μM) were: 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 indicate that ACE and NEP, two enzymes widely distributed in the body, may be involved in the metabolism of SP and NT.