Aminopeptidase P (APPro) is a manganese-containing enzyme that catalyses the
hydrolysis of the N-terminal residue of a polypeptide if the second residue is
proline. Structures of APPro mutants with reduced or negligible activity have
been determined in complex with the tripeptide substrate ValProLeu. In the
complex of Glu383Ala APPro with ValProLeu one of the two metal sites is only
partly occupied, indicating an essential role for Glu383 in metal binding in the
presence of substrate. His361Ala APPro clearly possesses residual activity as
the ValProLeu substrate has been cleaved in the crystals; difference electron
density consistent with bound ProLeu dipeptide and a disordered Val amino acid
is present at the active site. Contrary to previous suggestions, the His243Ala
mutant is capable of binding substrate. The structure of the His243Ala APPro
complex with ValProLeu shows that the peptide interacts with one of the
active-site metal atoms via its terminal amino group. The implications of these
complexes for the roles of the respective residues in APPro catalysis are
discussed.
