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Probing the Role of a “First Shell” Interaction in Trypsin‐Fold Serine Proteases
Author(s) -
Sharma Abriti,
Baird Teaster
Publication year - 2012
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.26.1_supplement.751.3
Subject(s) - trypsin , chemistry , proteases , hydrogen bond , stereochemistry , serine , substrate (aquarium) , hydrolase , binding site , amide , macromolecule , trypsin inhibitor , hydrophobic effect , enzyme , biochemistry , organic chemistry , molecule , biology , ecology
Several co‐crystal structures of Trypsin complexed with macromolecular inhibitors show that the backbone carbonyl oxygen of Phe 41 accepts a hydrogen from a backbone amide of inhibitor. This hydrogen bond interaction seems to be facilitated by the interaction between the phenyl ring of Phe 41 and methylene groups of Lys 60 . The variant Phe4 1 –>Ala (F41A‐Tg) was generated to probe the role and/ or contribution of position 41 in substrate/ inhibitor binding in S1′ pocket of Trypsin. The variant was constructed using PCR mutagenesis, expressed in P. Pastoris and purified using hydrophobic interaction chromatography. It was expected that the smaller Ala 41 would allow significant deviation from wild‐type structure and decrease the interaction with substrate/inhibitor resulting in weaker binding. The initial kinetic characterization suggest that the substitution does decrease the proteolytic activity of Trypsin and that the position may play an instrumental role in Trypsin activity. [This work was supported by Research Infrastructure at Minority Serving Institutions Fellowship, NIMHD Award P20MD000544 (AS)]