FIGURE 5.

Model of peptide substrate in the TPP1 active site. The model was constructed by docking a hexapeptide substrate, AAFFAA, with a fixed position of the carbonyl oxygen of the P1 residue (Phe) relative to the catalytic domain of TPP1, based on the prokumamolisin structure (PDB ID 1T1E) (28), onto the catalytic domain of TPP1 in which the side chains of active site residues Glu²⁷² and Asp²⁷⁶ were reoriented in the presumed active conformation as found in mature bacterial homologs. A ribbon diagram and a transparent surface rendition of the catalytic domain are shown in gray, a ball-and-stick depiction of the modeled hexapeptide substrate in yellow, and the superimposed linker with side chains of featured residues shown in ball-and-stick mode in cyan. Overlapping residues are shown in dotted surface. This model is an approximation because the positions of the mα3 helix with Glu²⁷² and Asp²⁷⁶ as well as mβ4 with Ser³²⁴ together with the mβ4-mβ5 loop with Asp³²⁷ are likely to differ in the mature enzyme (Fig. 4). The N terminus of the tripeptide substrate is likely to interact with the Asp³²⁷ side chain carboxyl groups. The predicted protonation state of Asp³²⁷ (see text) is consistent with a role in binding to an unsubstituted positively charged N terminus of the P3 residue of a putative substrate, thus providing a possible structural basis for the tripeptidyl peptidase activity. In addition, in the absence of crystal lattice contacts, positively charged Arg¹⁸³ of the linker is likely to participate in electrostatic interactions with negatively charged Asp³⁶⁰ of the oxyanion hole, potentially restricting its role in general acid and base catalysis.