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Kinetic studies on aminopeptidase M‐mediated degradation of human hemorphin LVV‐H7 and its N ‐terminally truncated products
Author(s) -
John Harald,
John Stefanie,
Forssmann WolfGeorg
Publication year - 2008
Publication title -
journal of peptide science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 66
eISSN - 1099-1387
pISSN - 1075-2617
DOI - 10.1002/psc.1002
Subject(s) - enzyme kinetics , aminopeptidase , chemistry , cleavage (geology) , enzyme , kinetics , in vivo , amino acid , stereochemistry , in vitro , ic50 , proteolysis , enkephalin , biochemistry , receptor , biology , leucine , active site , physics , paleontology , microbiology and biotechnology , quantum mechanics , fracture (geology) , opioid
The human hemorphin LVV‐H7 belongs to the class of µ‐opiod receptor‐binding peptides, which also exhibits significant affinity to insulin‐regulated aminopeptidase (IRAP) thereby affecting IRAP inhibition. The inhibitory potency towards IRAP is of pharmaceutical interest for the treatment of Alzheimer's disease. Consecutive N ‐terminal cleavage of the first two amino acid residues of LVV‐H7 affects a drastic increase of the binding affinity (V‐H7) but ultimately leads to its complete abolition after cleavage of the next amino acid residue (H7). Therefore, we investigated LVV‐H7 truncation by aminopeptidase M (AP‐M) identified as a LVV‐H7 degrading enzyme potentially regulating hemorphin activity towards IRAP in vivo . Using a selective quantitative multi‐component capillary zone electrophoretic method (CZE‐UV), we analyzed the AP‐M‐mediated subsequent proteolysis of the hemorphins LVV‐H7 (L 32 ‐F 41 ), VV‐H7 (V 33 ‐F 41 ), and V‐H7 (V 34 ‐F 41 ) in vitro . Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities ( V max ), catalytic constants (turnover numbers, k cat ), and specific enzyme activities (EA) were calculated. L 32 cleavage from LVV‐H7 happens more than two‐times faster ( k cat : 140 min −1 ± 9%, EA: 1.0 U/mg ± 9%) than V 33 cleavage from VV‐H7 ( k cat : 61 min −1 ± 10%, EA: 0.43 U/mg ± 10%) or V 32 deletion from V‐H7 ( k cat : 62 min −1 ± 8%, EA: 0.46 U/mg ± 8%). In contrast, we showed that H7 (Y 35 ‐F 41 ) was neither degraded by porcine AP‐M nor did it act as an inhibitor for this enzyme. Determined turnover numbers were in the same dimension as those reported for dynorphin degradation. This is the first time that AP‐M‐mediated truncation of natural underivatized LVV‐H7 and its physiological metabolites was analyzed to determine kinetic parameters useful for understanding hemorphin processing and designing hemorphin‐derived drug candidates. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.