Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2):: Insights into its mechanism of action

The molecular components ensuring the strict exopeptidase action of aminopeptidase N (APN) and related zinc aminopeptidases of the M-1 family have not yet been clearly established. The specific recognition of the N-terminal amino acid of the substrates by the enzymes has been proposed to involve either the complexation of the free amino group by the catalytic zinc ion or an interaction with an anionic binding site, which could be constituted by an aspartate or glutamate residue. To investigate the existence of such an ionic binding site, site-directed mutagenesis experiments have been performed on acidic residues of pig APN. Given that aminopeptidases of the M-1 family are likely to have a common mechanism of action, only strictly conserved residues were mutated. As compared to the wild-type enzyme, the mutation (DE)-E-220 led only to slight modifications in the kinetic parameters of the enzyme and in the K-i, values of various inhibitors, indicating that this residue is not critically involved in the hydrolytic mechanism. In contrast, the mutations E(350)Q and (ED)-D-350 induced a large decrease in enzyme activity, essentially due to modifications in k(cat), whereas the E(350)A mutation led to an almost completely inactive enzyme. Moreover, among the inhibitors tested, only those acting as transition state analogs showed significant increases in their Ki values. These data are in favor of E-350 belonging to the "anionic binding site" in APN. A mechanism of action, derived from that of thermolysin, is proposed for these aminopeptidases, which explains the importance of E-350 in transition stale formation, rather than in the Michaelis complex.