Premium
Engineering trypsin‐fold serine proteases for inhibitor resistance
Author(s) -
Batt Anna,
Baird Teaster
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.561.12
Subject(s) - chemistry , trypsin , macromolecule , hydrogen bond , peptide , stereochemistry , cyclic peptide , peptide bond , trypsinogen , serine , proteases , biochemistry , enzyme , molecule , organic chemistry
In several co‐crystal structures of trypsin and its macromolecular inhibitors, the hydroxyl group of Tyr 39 and backbone carbonyl of Phe 41 of trypsin lie within hydrogen bonding distance of the P4′ and the P2′ backbone amides of the inhibitor, respectively. Lys 60 and Tyr 39 form a “cage” around Phe 41 , likely keeping the backbone carbonyl of Phe 41 optimally positioned for the hydrogen bond at P2′. Several trypsinogen variants, in which various residues within the prime‐side binding interface were replaced with a range of alternate amino acid residues, were constructed to evaluate how each of the prime‐side trypsin residues affect macromolecular inhibitor binding. It is hypothesized that disruption of the hydrogen bonds at P2′ and P4′ will allow more conformational freedom and thereby weaken macromolecular inhibitor binding. Initial kinetic characterization and inhibition studies of the variants have been carried out using small commercially available peptide substrates (1–3 residues), and several macromolecular inhibitors. Preliminary data suggest that the constructed variants and wild‐type trypsin show similar affinities for the small peptide substrates (K M = 26 ± 3 μM), while affinity for macromolecular inhibitors appears to vary for the variants relative to wild‐type. This work was supported by NSF CAREER Award MCB‐0643988–02 and NIMHD Award P20MD000544.