Characterization of the thermolysin-like cleavage of biologically active peptides by Xenopus laevis peptide hormone inactivating enzyme

Abstract

Peptide hormone inactivating endopeptidase (PHIE) is a metalloendopeptidase which was isolated from the skin granular gland secretions of Xenopus laevis [Carvalho, K. M., Joudiou, C., Boussetta, H., Leseney, A. M., & Cohen, P. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 84-88]. This peptidase exhibits a thermolysin-like character and hydrolyzes bonds on the amino terminus of hydrophobic amino acids, performing cleavage of Xaa-Phe, Xaa-Leu, Xaa-Ile, Xaa-Tyr, and Xaa-Trp doublets. When the enzyme recognized a doublet of hydrophobic amino acids such as Phe6-Phe7 of somatostatin-14, Phe7-Phe8 of substance P, Phe4-Leu5 of [Leu5,Arg6]enkephalin, and Tyr4-Ile5 of angiotensin II, cleavage occurred preferentially between these residues. The use of selectively modified carboxy-terminal octapeptide fragments of atrial natriuretic factor (ANF) indicated that the enzyme tolerates as substrates only peptides bearing a P'1 bulky hydrophobic amino acid residue. Although a P'1 hydrophobic residue was a necessary condition, it was found in a number of peptides that all potential cleavage sites were not recognized by the enzyme. These data suggested that this metalloendoprotease requires for its thermolysin-like activity a preferred conformation of the peptide chain. Kinetic results obtained using a series of related substrates derived from biologically active peptides of the atrial natriuretic factor, tachykinin, and enkephalin families indicated the presence of an extended binding site accommodating at least six amino acid residues, in contrast to thermolysin (EC 3.4.24.4) and neutral endopeptidase (NEP; EC 3.4.24.11), which hydrolyze shorter homologous peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide Substrate Specificity and Properties of the zinc‐endopeptidase Activity of Botulinum Type B Neurotoxin

“…There is growing evidence that a degree of substrate secondary structure is required for optimal activity in other zinc metalloproteases. Zinc endopeptidases isolated from rat testes and the skin secretion of Xenopus laevis have both been shown to display higher affinity binding to larger peptide substrates (Chesneau et al, 1994;Joudiou et al, 1993). The processing of the peptide hormone precursor prosomatostatin also appears to be enhanced by the presence of secondary structure within the polypeptide chain (Brakch et al, 1993).…”

Purification and Characterization of a Vasoactive Intestinal Polypeptide-degrading Endoprotease from Porcine Antral Mucosal Membranes

“…Rapid and specific inactivation of a neuropeptide after its function is important and is thought to depend on the action of its inactivating enzymets). There have been many reports of different peptidases which may be involved in neuropeptide degradation (16,(26)(27)(28)(29)(30)(31)(32), but there is not much convincing evidence as yet for any of them that it is specific for a single peptide. In the present study, we have attempted to isolate a novel protease(s) from porcine antral mucosa which may be involved in neuropeptide processing or degradation using a synthetic peptide MCA substrate, Boc-Arg-Val-Arg-Arg-MCA, and could finally isolate a 37,000 dalton neutral endoprotease, which was found to cleave VIP highly efficiently as well as specifically.…”

Conformational Study Based on Simulated Annealing and1H NMR of Peptides Comprising the Consensus Sequence Arg5-Asp-Val-Arg-Gly9: Effects of the Substitution Ser 12 by Ala 12 or of 3 Residues Deletion at the N-Terminus

“…About the implication of these structural differences on the biological activity, one should notice that the [4][5][6][7][8][9][10][11] 667 Simulated Annealing and 1 H NMR of Peptides should therefore facilitate the the recognition of the undecapeptide (hence the enhanced activity), while the tetradecapeptide structurales to an hairpin-like conc formation in the presence of the enzyme which perhaps modifies its electrostatic equilibrium. Table VII gives the individual rms differences over the heavy backbone atoms, between the lowest energy structures of both classes # 1 and #2 of both the tetradecapeptide and the undecapeptide, taken two by two, when fitting over residues 4 to 9.…”