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Introducing extended substrate selectivity into trypsin through redesign
Author(s) -
Najibi Sayeeda Phyllissity,
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.837.4
Subject(s) - selectivity , chemistry , trypsin , serine , hydrolase , hydrolysis , substrate (aquarium) , peptide , enzyme , catalysis , biochemistry , biology , ecology
The objective of this research was to evaluate how a residue with hydrogen bond capability may affect substrate selection by a previously engineered threonine protease. C42S/C58V/S195T trypsin (SVT‐Tn) was characterized and compared to trypsin variants C42A/C58V/S195T‐Tn (AVT‐Tn) and C42A/C58A/S195T (AAT‐Tn). p ‐Nitroanilide (pNA) and 7‐amino 4‐methyl coumarin (AMC) derivatives of benzyloxycarbonyl‐Gly‐Pro‐Arg (Z‐GPR) were use to kinetically evaluate the activity and specificity of SVT‐Tn. Their respective k cat /K M values show that SVT‐Tn is 8‐ and 40‐fold more efficient in Z‐GPR‐pNA and Z‐GPR‐AMC hydrolysis, respectively, than AVT‐Tn. Additionally, SVT‐Tn was 5‐fold less efficient in Z‐GPR‐pNA hydrolysis and 2‐fold more efficient in Z‐GPR‐AMC hydrolysis than AAT‐Tn. However, the (k cat /K M ) pNA /(k cat /K M ) AMC ratios show that SVT trypsin is 3.5‐fold more selective for the pNA substrate, less discriminatory than the AVT and AAT variants. The results suggest that a serine at position 42 in the S195T/C58V context significantly affects catalytic efficiency in a substrate dependent manner for the two substrates tested, but selectivity between the two substrates was only moderate. However, longer peptide substrates are needed to thoroughly evaluate the degree of the selectivity of SVT‐Tn. (This work was supported by NSF Award MCB‐0643988‐02)