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Enhancing the catalytic activity of an engineered threonine protease
Author(s) -
Lansang Mie,
Baird Teaster
Publication year - 2010
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.24.1_supplement.837.3
Subject(s) - trypsin , chemistry , thermostability , protease , threonine , enzyme , stereochemistry , biochemistry , valine , amino acid , serine
In this study, the Cys‐42—Cys‐58 disulfide bridge of S195T trypsin was replaced with two valine residues to: 1) increase threonine protease activity and 2) better understand the structual limitations and requirements of this subsite with respect to catalysis. The activities of C42V/C58V and C42V/C58V/S195T trypsins (VV‐Tn and VVT‐Tn, respectively) were evaluated with benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) p ‐nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) substrates. VVT‐Tn was 150 and 10 times more active towards the pNA and AMC substrates, respectively, than C42A/C58V/S195T trypsin (AVT‐Tn; k cat /K M values). In a thermostability study, both variants and WT trypsin retained > 80% of their activity suggesting that the substitutions did not destabilize the enzymes. The pH dependence of activity profiles were identical to WT trypsin, suggesting that any structural differences do not perturb the pK a s of catalytically relevant residues. Finally, the K I of p ‐aminobenzamidine was identical for native and VV‐Tn but that of VVT‐Tn was half the K I for WT. Therefore, Thr‐195 may affect the primary binding pocket structure. Collectively, these results indicate that the activity of Thr‐195 trypsin variants may be increased by appropriate substitutions at positions 42 and 58. However, the structural pertubations induced by these substitutions make achievement of wild‐type levels of activity less likely. (This work was supported by NSF Award MCB‐0643988‐02)