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

Abstract : 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 (Y-281, Y-229, Y-414, and Y-441) and one partially (Y-409) 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 (Y-414), in substrate specificity and/or catalytic efficiency (Y-409), in stabilization of essential amino acids of the active site (Y-229, Y-409) and potentially in the maintenance of its structural integrity (Y-281, Y-441). 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.
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Sandrine Cadel, Cecile Darmon, Julien Pernier, Guy Hervé, Thierry Foulon. The M1 family of vertebrate aminopeptidases: Role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B. Biochimie, Elsevier, 2015, 109, pp.67-77. ⟨10.1016/j.biochi.2014.12.009⟩. ⟨hal-01558872⟩

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