The KEEN interaction interface. A, crystal structure of PPEP-1(E143A/Y178F) in complex with PP-pep with detail of the KEEN interaction interface. The substrate peptide (PP-pep) is colored and labeled in blue. PPEP-1 components of the KEEN interface (Lys-101, orange; Glu-184 and Glu-185, green) and metal ion–coordinating histidine residues (His-142 and His-146; gray) are shown as sticks. PPEP-1 components are labeled black. Hydrogen bonds are indicated as dotted lines (PDB code 5A0X). B, specific activity of WT PPEP-1 and KEEN interaction interface mutants in the presence of Abz-PP-Dnp, Abz-AP-Dnp, or Abz-PA-Dnp. The measurements were carried out in triplicate. Error bars are the S.D. with average values above the bars. C, Michaelis–Menten parameters of PPEP-1(E184A) for substrate Abz-PP-Dnp. The same PPEP-1(WT) data are shown as in . D, the KEEN interaction interface is disrupted by the E184A mutation but not by the absence of Asn-3* in the substrate peptide. The KEEN interaction interface of both cocrystal structures with PP-pep (left) or APP-pep (right) is shown. The substrate peptide is colored in blue, the S-loop is in yellow, the active-site helix is in orange, and the gluzincin characteristic helix is in pink. The electron density (2mFo − DFc) around the substrate peptide, catalytic Zn2+ ions, and the side chains of Lys-101, His-142, His-146, Ala-184, and Glu-185 is shown at a contour level of 1 r.m.s.d. (0.31 e/Å3). The difference electron density (mFo − DFc) is shown as green mesh with a contour level of +3 r.m.s.d. (0.31 e/Å3).