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

Abstract

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 Zn 2+-metallopeptidases that are characterized by two highly conserved motives, GXMEN (potential substrate binding site) and HEXXHX 18E (Zn 2+-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 G 298XM 300E 301N 302 motif and one mutant of the HEIS 328HX 18E motif were expressed in Escherichia coli. All mutations except G 298P, G 298S, and S 328A abolished the aminopeptidase activity. The S 328A mutant mimics the sequence of bovine Ap-B Zn 2+-binding site, which differs from those of other mammalian Ap-B. This mutant conserved a canonical Ap-B activity. G 298S and G 298P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl − anions. Moreover, the G 298P 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 G 298 plays in the catalytic mechanism of Ap-B. Our results show that G 298 is essential to Ap-B activity and participates to the substrate specificity of the enzyme.