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(18)E (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(298)XM(300)E(301)N(302) motif and one mutant of the HEIS(328)HX(18)E motif were expressed in Escherichia coli. All mutations except G(298)P, G(298)S, and S(328)A abolished the aminopeptidase activity. The S(328)A 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(298)S and G(298)P mutants exhibit new enzymatic properties such as changes in their profile of inhibition and their sensitivity to Cl(-) anions. Moreover, the G(298)P 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.