Engineering trypsin for development of inhibitor resistance

An efficient protease therapeutic must be more resistant to naturally occurring inhibitors compared to wild type, while maintaining its catalytic activity. A key step in development of such therapeutics is identifying the residues that are involved in protease-inhibitor interactions. In trypsin, the backbone carbonyl oxygen of phenylalanine 41 (F41) and hydroxyl group of tyrosine 39 (Y39) interact with the backbone amide of different macromolecular inhibitors at positions P2' and P4' respectively, suggesting that these may be key residues in trypsin-inhibitor interactions. To test this hypothesis, several trypsin (Tn) variants (F41A, Y39A, F41G, Y39A/F41G, Y39G/F41G, Y39A/F41P, Y39G/F41P) were constructed. Due to time limitation and complications in the project, only two variants F41A and Y39A/F41A were characterized. As expected, F41A showed similar catalytic activity compared to wild type (WT) whereas the double variant Y39A/F41A demonstrated significantly lower catalytic activity. Km values for WT, F41A and Y39A/F41A were (7.0 ± 0.6) pM, (11.1 ± 1.7) pM and (5.7 ± 0.1) pM respectively. Catalytic efficiency (kcat/KM) for WT, F41A and Y39A/F41A was (9.6 ± 0.8) x 108s-1M-1, (2.6 ± 0.4) x 109s-1M-1 and (7.1 ± 0.2) x 106s-1M-1 respectively. Inhibition studies suggested that F41A was more resistant to macromolecular inhibitors such as BPTI and SBTI compared to wild type. At a 1:3 molar ratio of enzyme to BPTI, wild type retained only 2% of its activity whereas F41A retained 17% of its activity. Similarly, at a 1:5 molar ratio of enzyme to SBTI, wild type retained 10% activity whereas F41A was able to retain 25% activity. Inhibition data for the double variant, Y39A/F41A, however, were not conclusive.