Additivity-based design of the strongest possible turkey ovomucoid third domain inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB)

Abstract

We describe here successful designs of strong inhibitors for porcine pancreatic elastase (PPE) and Streptomyces griseus protease B (SGPB). For each enzyme two inhibitor variants were designed. In one, the reactive site residue (position 18) was retained and the best residues were substituted at contact positions 13, 14, and 15. In the other variant the best residues were substituted at all contact positions except the reactive site where a Gly was substituted. The four designed variants were: for PPE, T(13)E(14)Y(15)-OMTKY3 and T(13)E(14)Y(15)G(18)M(21)P(32)V(36)-OMTKY3, and for SGPB, S(13)D(14)Y(15)-OMTKY3 and S(13)D(14)Y(15)G(18)I(19)K(21)-OMTKY3. The free energies of association (ΔG(0)) of expressed variants have been measured with the proteases for which they were designed as well as with five other serine proteases and the results are discussed.

Keywords: Additivity; CARL; HLE; Inhibitor design; Kazal inhibitor; OMTKY3; PPE; Protease inhibitor; SGPA and SGPB; SRA; Serine protease; Streptomyces griseus protease A and B. In MEROPS database and recent literature these are listed as Streptogrisin A and B; human leukocyte elastase; porcine pancreatic elastase; sequence to reactivity algorithm; subtilisin Carlsberg; turkey ovomucoid third domain.

Figure 1

Primary structure of expressed wtOMTKY3. The first five residues are not shown because they are not part of the expressed OMTKY3 (and its variants) and have been found to have no effect on the inhibitory activity of OMTKY3. The residues are numbered sequentially as well as in Schechter-Berger notation [30]. In Schechter-Berger notation, the reactive site residue (shown by the arrow) is labeled as P₁. Residues towards the N-terminal of the P₁ residue are sequentially labeled as P₂, P₃, …P_n whereas residues C-terminal to P₁ residue are labeled as P₁', P₂', … P_n'. The amino acids shown as filled circles are the consensus contact residues in inhibitor-protease complexes. The two filled circles shown in grey color represent the residues which in addition to being the contact residues also play a structural role.