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Substrate specificity of rabbit liver metalloendopeptidase and its new fluorogenic peptide substrates
N Kojima1, S Kawabata, Y Makinose
1Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Abstract:
A metalloendopeptidase (MEP) isolated from rabbit liver microsomes with substrate specificity for peptides containing Arg at the P1 and P4 positions has recently proved to be identical to soluble angiotensin-binding protein present in the cytosol. Here we describe the peptide-degrading specificity of MEP, determined using various bioactive peptides and novel fluorogenic substrates for the enzyme. MEP degraded oligopeptides, including bradykinin, alpha-neoendorphin, bovine adrenal medulla dodecapeptide, substance P, bombesin, neurotensin, and alpha-endorphin, but not polypeptides such as reduced lysozyme and histone H4, hence, MEP probably belongs to the family of endo-oligopeptidases. It cleaved most preferentially at the -Phe-Ser- bond of bradykinin (kcat/Km = 2.8 x 10(4) M-1.S-1) but did not cleave high molecular weight and low molecular weight kininogens, the precursors of bradykinin. MEP did not cleave angiotensin I, dynorphin A 1-13, somatostatin, and luteinizing hormone-releasing hormone, some of which are good substrates for metalloendopeptidase-24.15, metalloendopeptidase-24.16, N-arginine dibasic convertase, and yeast endopeptidase-24.15 related peptidase. An active site-directed inhibitor of metalloendopeptidase-24.15, N-[1-(R,S)-carboxyl-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate also had no effects on the amidolytic activity of MEP. Based on the cleavage sites of bioactive peptides and processing sites of vitamin K-dependent proproteins, intramolecularly quenched fluorogenic peptide substrates were newly synthesized. Among the thirteen substrates used, the most reactive was 2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala- Asn-Ser-2,4-dinitroanilinoethylamide (kcat/Km = 9.3 x 10(5) M-1.S-1). An angiotensin antagonist, [Sar1, Ala8]-angiotensin II, inhibited hydrolysis of the substrate by MEP in a competitive manner (Kl = 7.6 microM). MEP cleaved oligopeptides even on the carboxyl side of proline residue and these peptides are resistant to hydrolysis by the cytosol-derived proteasome, therefore MEP may participate in the catabolism of oligopeptides in the cytosol, together with other endo-oligopeptidases.
Insights
Metalloendopeptidase (MEP) degrades various bioactive oligopeptides, including bradykinin, but not larger polypeptides. This enzyme, also known as soluble angiotensin-binding protein, may play a role in cytosolic oligopeptide catabolism.
Area of Science:
- Biochemistry
- Enzymology
Background:
- A metalloendopeptidase (MEP) from rabbit liver microsomes, specific for Arg at P1 and P4 positions, is identical to soluble angiotensin-binding protein.
- MEP's role in peptide degradation and its relationship to other peptidases are not fully elucidated.
Purpose of the Study:
- To characterize the peptide-degrading specificity of MEP.
- To identify novel substrates and inhibitors for MEP.
- To elucidate MEP's potential role in cytosolic oligopeptide metabolism.
Main Methods:
- Degradation assays using various bioactive peptides (bradykinin, substance P, endorphins, etc.).
- Synthesis and use of novel fluorogenic peptide substrates designed based on cleavage sites.
- Enzyme kinetics analysis (kcat/Km) and inhibition studies with an angiotensin antagonist.
Main Results:
- MEP degraded various bioactive oligopeptides but not polypeptides like lysozyme or histone H4, suggesting it is an endo-oligopeptidase.
- Preferential cleavage of bradykinin at the Phe-Ser bond was observed (kcat/Km = 2.8 x 10(4) M-1.S-1).
- A novel fluorogenic substrate (2-aminobenzoyl-Ala-Arg-Val-Arg-Arg-Ala-Asn-Ser-2,4-dinitroanilinoethylamide) showed high reactivity (kcat/Km = 9.3 x 10(5) M-1.S-1), competitively inhibited by an angiotensin antagonist.
Conclusions:
- MEP functions as an endo-oligopeptidase, degrading specific bioactive oligopeptides in the cytosol.
- Its ability to cleave peptides resistant to proteasomal degradation suggests a distinct role in peptide metabolism.
- MEP's specificity and activity profile differentiate it from other known metalloendopeptidases.