Premium
Hydrohpobic interactions in an engineered threonine protease
Author(s) -
Lansang Mie,
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.517.4
Subject(s) - trypsin , chemistry , protease , context (archaeology) , threonine , substrate (aquarium) , enzyme , biochemistry , active site , binding site , hydrolysis , stereochemistry , biology , paleontology , ecology , serine
In an effort to understand the contributions of hydrophobicity and geometry in the activity of an engineered threonine protease, cysteine residues 42 and 58 were substituted with alanines and/or valines in the Ser‐195→Thr background trypsin. Four trypsin variants [C42A/C58A/S195T (AAT‐Tn) C42A/C58V/S195T (AVT‐Tn), C42V/C58V/S195T (VVT‐Tn) and, C42V/C58A/S195T (VAT‐Tn)] were constructed, expressed and purified. Their catalytic properties were evaluated using the substrate N‐α‐benzyloxycarbonyl‐Gly‐Pro‐Arg p ‐nitroanilide. Compared to wild‐type trypsin, AAT‐Tn and AVT‐Tn retained ≤ 0.3% of activity while VVT‐Tn and VAT‐Tn variants retained only 0.7% and 0.1% wild‐type activity, respectively. The trend in the data suggests that increased hydrophobicity in the altered site leads to increased hydrolytic activity in the Ser‐195→Thr context. Interestingly, all four of the trypsin variants were inhibited by p ‐aminobenzamidine, a competitive inhibitor of trypsin, more strongly than wild type, suggesting that the substitutions also induce a structural change in primary substrate binding site. The increased activity with increased hydrophobicity is likely due to favorable interaction with the methyl group of Thr‐195. However, this interaction may lead to structural pertubations in the primary binding site. Further experiments are needed to confirm these hypotheses. (This work was supported by NSF Award MCB‐0643988‐02)