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.

Aminopeptidase B (Ap-B) is a Zn 2þ-aminopeptidase of the M1 family which is implicated, in conjunction with the nardilysin endoprotease, in the generation of miniglucagon, a peptide involved in the maintenance of glucose homeostasis. Other in vivo physiological roles have been established for this vertebrate enzyme, such as the processing of Arg-extended forms of human insulin and cholecystokinin 9 and the degradation of viral epitopes in the cytoplasm. Among M1 family members, Ap-B is phylogenetically close to leukotriene A 4 hydrolase (LTA 4 H), a bi-functional aminopeptidase also able to transform LTA 4 in LTB 4 (a lipid mediator of inflammation). As the activities of LTA 4 H are reported to be inhibited by resveratrol, a polyphenolic molecule from red wine, the effect of this molecule was investigated on the Ap-B activity. Several other active phenolic compounds produced in plants were also tested. Among them, curcumin and mangiferin are the most effective inhibitors. Dixon analysis indicates that curcumin is a non-competitive inhibitor with a Ki value of 46 mmol.L À1. Dixon and Lineweaver-Burk representations with mangiferin show a mixed non-competitive inhibition with Ki' and Ki values of 194 mmol.L À1 and 105 mmol.L À1 , respectively. At 200 mmol.L À1 , no significant effect was observed with caffeic, chlorogenic, ferulic, salicylic and sinapic acids as well as with resveratrol. Analyses on the 3D-structure of LTA 4 H with resveratrol (pdb: 3FTS) and the Ap-B 3D-model allow hypothesis to explain theses results.