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

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, T¹³E¹⁴Y¹⁵-OMTKY3 and T¹³E¹⁴Y¹⁵G¹⁸M²¹P³²V³⁶-OMTKY3, and for SGPB, S¹³D¹⁴Y¹⁵-OMTKY3 and S¹³D¹⁴Y¹⁵G¹⁸I¹⁹K²¹-OMTKY3. The free energies of association (ΔG⁰) 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.