The M1 family of vertebrate aminopeptidases: Role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B

Aminopeptidase B (Ap-B), a member of the M1 family of Zn2+-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Biochimie 2015-02, Vol.109, p.67-77
Hauptverfasser: Cadel, Sandrine, Darmon, Cécile, Pernier, Julien, Hervé, Guy, Foulon, Thierry
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung:Aminopeptidase B (Ap-B), a member of the M1 family of Zn2+-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases. [Display omitted] •Y229 appears to be a crucial actor in the catalytic mechanism.•Y229H substitution preserves the activity and catalytic efficiency.•Y229 hydroxyl stabilizes E301, an essential amino acid of the active site.•The conserved tyrosines of the M1 family, even distant from the active site, are essential for the enzymatic activity.•The inhibitory effect of arphamenine A or B is dependent of the Y409 residue.
ISSN:0300-9084
1638-6183
DOI:10.1016/j.biochi.2014.12.009