A glutamate residue contributes to the exopeptidase specificity in aminopeptidase A

Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound aminopeptidase that contains the consensus sequence HEXXH (385-389) found in the zinc metalloprotease family, the zincins. Sequence alignment of the mouse APA with other monozinc-aminopeptidases indicates the presence of a highly conserved glutamate residue (Glu(352) in, APA) found in the conserved motif GAMEN (349-353). In monozinc-aminopeptidases, the negative charge of the glutamate side-chain carboxylate may constitute the anionic binding site involved in the recognition of the free amino group of substrates or inhibitors. The functional role of Glu(352) in, APA was investigated by substituting this residue with an aspartate (Asp(352)), a glycine (Gly(352)),a glutamine (Gln(352)) or an arginine (Arg(352)) residue by site-directed mutagenesis. Kinetic studies showed that the K-m, values of the mutant enzymes were unaffected, whereas k(cat) values were decreased 10-250-fold, resulting in a 10-, 30- 260- and 400-fold reduction in cleavage efficiencies for the mutants Asp(352), Gly(352), Gln(352) and Arg(352) respectively. The inhibitory potency of two different classes of inhibitors, a thiol and a phosphonate compound, was significantly (P < 0.05) decreased by 10- and 4-fold respectively in the mutated enzymes. Moreover, the inhibitory potency of angiotensin I, used as a competitor of the synthetic substrate alpha-L-glutamyl beta-naphthylamide, displayed a 4-fold reduction (P < 0.01) in the mutated enzymes, whereas the K-i values of its N-acetyl derivative were unchanged. These data strongly suggest that Glu(352) is involved in the catalytic process of APA and contributes to the exopeptidase activity of this enzyme through interaction with the N-terminal part of substrates or inhibitors.