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The role of a second shell hydrophobic interaction in trypsin‐fold serine protease function
Author(s) -
Batt Anna,
Baird Teaster
Publication year - 2011
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.25.1_supplement.517.1
Subject(s) - chemistry , trypsin , hydrogen bond , stereochemistry , macromolecule , peptide bond , alanine , cleavage (geology) , scissile bond , protease , amide , hydrophobic effect , active site , serine protease , binding site , hydrolysis , peptide , enzyme , amino acid , biochemistry , molecule , organic chemistry , geotechnical engineering , fracture (geology) , engineering
In several co‐crystal structures of trypsin and its macromolecular inhibitors, the backbone carbonyl of Phe 41 accepts a hydrogen bond from the P2′ backbone amide in the inhibitor (a “first shell” interaction) suggesting that this hydrogen bond interaction is significant. Lys 60 and Tyr 39 form a “cage” around Phe 41 (a “second shell” interaction), likely keeping its backbone carbonyl optimally positioned for hydrogen bonding. A variant, in which Tyr 39 was replaced with an alanine (Y39A‐Tn), was constructed to evaluate how removal of the cage around Phe 41 affects activity. It is hypothesized that the removal will allow Phe 41 more conformational freedom and thereby weaken substrate/inhibitor binding. The Y39A‐Tn variant has been constructed using PCR mutagenesis, expressed in P. pastoris and purified using hydrophobic chromatography. Initial kinetic characterization will be carried out using commercially available, small (1–3 residues) peptide substrates. Given the location and proposed role of Phe 41 , and that the active site of the enzyme has not been modified, the substitution is expected to effect little change on the hydrolytic activity of said substrates. However, notable changes in cleavage of proteins and decreased affinity of macromolecular substrates are expected. (This work was supported by NSF Award MCB‐0643988‐02)