Mutation in the substrate-binding site of aminopeptidase B confers new enzymatic properties

Aminopeptidase B (Ap-B) catalyzes the cleavage of arginine and lysine residues at the N-terminus of various peptide substrates. In vivo, it participates notably in the miniglucagon and cholecystokinin 8 processing, but the complete range of physiological functions of Ap-B remains to be discovered. Ap-B is a member of the M1 family of Zn2+-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX18E (Zn2+-binding site). In this study, mutagenesis and molecular modelling were used to investigate the enzymatic mechanism of Ap-B. Nineteen rat Ap-B mutants of the G298XM300E301N302 motif and one mutant of the HEIS328HX18E motif were expressed in Escherichia coli. All mutations except G298P, G298S, and S328A abolished the aminopeptidase activity. The S328A mutant mimics the sequence of bovine Ap-B Zn2+-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G298S and G298P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl− anions. Moreover, the G298P mutant exhibits new substrate specificity. A structural analysis using circular dichroism, fluorescence spectroscopy, molecular modelling and dynamics was performed to investigate the role that residue G298 plays in the catalytic mechanism of Ap-B. Our results show that G298 is essential to Ap-B activity and participates to the substrate specificity of the enzyme.

Research highlights
► GXMEN motif drives Ap-B activity.
► G298 binds substrates and inhibitors.
► G298P mutation reorganizes Ap-B catalytic site.
► G298P mutation abolishes Ap-B substrate specificity.