title: 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 Zn(2+)-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.
authors.affiliation: Sorbonne universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), FR 3631 UPMC-CNRS, BIOSIPE, BIOgenèse des SIgnaux PEptidiques, Paris, France. Electronic address: marie-sandrine.cadel@upmc.fr.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Cadel
authors.affiliation: Sorbonne universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), FR 3631 UPMC-CNRS, BIOSIPE, BIOgenèse des SIgnaux PEptidiques, Paris, France. Electronic address: cecile.darmon@upmc.fr.
authors.forename: Cécile
authors.initials: C
authors.lastname: Darmon
authors.affiliation: Cytoskeleton Dynamics and Motility Group, Laboratoire d'Enzymologie et Biochimie Structurale [LEBS], UPR 3082 - CNRS, Gif-sur-Yvette, France. Electronic address: pernier@lebs.cnrs-gif.fr.
authors.forename: Julien
authors.initials: J
authors.lastname: Pernier
authors.affiliation: Sorbonne universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), FR 3631 UPMC-CNRS, BIOSIPE, BIOgenèse des SIgnaux PEptidiques, Paris, France. Electronic address: guy.herve@upmc.fr.
authors.forename: Guy
authors.initials: G
authors.lastname: Hervé
authors.affiliation: Sorbonne universités, UPMC Univ Paris 06, Institut de Biologie Paris-Seine (IBPS), FR 3631 UPMC-CNRS, BIOSIPE, BIOgenèse des SIgnaux PEptidiques, Paris, France. Electronic address: thierry.foulon@upmc.fr.
authors.forename: Thierry
authors.initials: T
authors.lastname: Foulon
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D055162:Biocatalysis; D015153:Blotting, Western; D017124:Conserved Sequence / Q000235:genetics*; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D019143:Evolution, Molecular; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014714:Vertebrates / Q000235:genetics* / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D014443:Tyrosine; D004743:Enkephalin, Leucine; C026768:enkephalin-Leu, Arg(6)-; D000626:Aminopeptidases; C019721:aminopeptidase B
keywords: Aminopeptidases; Leukotriene A(4) hydrolase; M1 family; Tyrosine; Zn(2+)-metallopeptidase