Media type: E-Article Title: Kinetic studies on aminopeptidase M‐mediated degradation of human hemorphin LVV‐H7 and its N‐terminally truncated products Contributor: John, Harald; John, Stefanie; Forssmann, Wolf‐Georg imprint: Wiley, 2008 Published in: Journal of Peptide Science Language: English DOI: 10.1002/psc.1002 ISSN: 1075-2617; 1099-1387 Keywords: Organic Chemistry ; Drug Discovery ; Pharmacology ; Molecular Biology ; Molecular Medicine ; General Medicine ; Biochemistry ; Structural Biology Origination: Footnote: Description: <jats:title>Abstract</jats:title><jats:p>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 <jats:italic>N</jats:italic>‐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 <jats:italic>in vivo</jats:italic>. 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<jats:sup>32</jats:sup>‐F<jats:sup>41</jats:sup>), VV‐H7 (V<jats:sup>33</jats:sup>‐F<jats:sup>41</jats:sup>), and V‐H7 (V<jats:sup>34</jats:sup>‐F<jats:sup>41</jats:sup>) <jats:italic>in vitro</jats:italic>. Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities (<jats:italic>V</jats:italic><jats:sub>max</jats:sub>), catalytic constants (turnover numbers, <jats:italic>k</jats:italic><jats:sub>cat</jats:sub>), and specific enzyme activities (EA) were calculated. L<jats:sup>32</jats:sup> cleavage from LVV‐H7 happens more than two‐times faster (<jats:italic>k</jats:italic><jats:sub>cat</jats:sub>: 140 min<jats:sup>−1</jats:sup> ± 9%, EA: 1.0 U/mg ± 9%) than V<jats:sup>33</jats:sup> cleavage from VV‐H7 (<jats:italic>k</jats:italic><jats:sub>cat</jats:sub>: 61 min<jats:sup>−1</jats:sup> ± 10%, EA: 0.43 U/mg ± 10%) or V<jats:sup>32</jats:sup> deletion from V‐H7 (<jats:italic>k</jats:italic><jats:sub>cat</jats:sub>: 62 min<jats:sup>−1</jats:sup> ± 8%, EA: 0.46 U/mg ± 8%). In contrast, we showed that H7 (Y<jats:sup>35</jats:sup>‐F<jats:sup>41</jats:sup>) 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.</jats:p>