Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A

Aminopeptidase A (EC 3.4.11.7, APA) is a homodimeric membrane-bound glycoprotein that contains the consensus sequence HEXXH (385-389) found in zinc metallopeptidases such as thermolysin, The x-ray structure of the latter enzyme revealed that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis, Alignment of the sequence of mouse APA with those of the already characterized metallopeptidases showed the presence of several conserved amino acids such as a glutamate residue in position 408 which may constitute the putative third zinc ligand. The functional implication of this residue and the role of glutamate 386 in the HELVH (385-389) motif of APA have been investigated by replacing these residues with an aspartate (Asp-386, Asp-408) or an alanine (Ala-386, Ala-408) by site-directed mutagenesis. Expressed mutated proteins in position 386 showed no APA activity, Ala-408 was also inactive, and Asp-408 had 5% of the wild type enzyme activity and a similar K-m. Zn-65 incorporation measurements indicated that Ala-386 binds the zinc ion as well as the wild type enzyme, whereas the Ala-408 mutant did not, These results provide evidence that Glu-408 is the third zinc-coordinating residue of APA, confirm the presumed involvement of Glu-386 in the catalytic process of the enzyme, and identify APA as a zinc metallopeptidase functionally similar to thermolysin.