Chymotrypsin is a biochemically well characterized serine endopeptidase that cleaves after amino acids with bulky hydrophobic side chains. In spite of its superimposable tertiary structure, its substrate specificity is very distinct from trypsin and elastase which cleave after amino acids with basic groups and small aliphatic side chains, respectively. Apart from high activity (kcat/KM = 106 M-1S-1, AAPF-AMC substrate), chymotrypsin also displays strong discrimination against non-cognate substrates (kcat/KM = 103 M-1S-1, AAPK-AMC substrate). This enigma of similarity in structure of enzymes with antonymous specificities has led to many rational engineering attempts to graft substrate specificity loops of other enzymes into chymotrypsin but only with little success.In the present work, we have taken a directed evolution approach to engineer the substrate binding pocket of chymotrypsin B. A platform using FACS for functional screening of large libraries (> 106 variants) based on autotransporter mediated surface display of chymotrypsin and fluorescent labeling to monitor proteolytic activity using peptide substrates containing a proteolysis-sensitive linker sandwiched between a FRET pair was developed. Multiple libraries including site saturation of specific residues in the binding pocket and error-prone PCR were screened by simultaneous selection for activity against desired substrate and counter-selection against wild type-like activity for isolating variants of chymotrypsin B specifically active against post translationally modified tyrosine residues. By assessing the residue changes in substrate binding pocket that confer new specificity, we gain insights for understanding the molecular basis of substrate discrimination in enzyme catalysis. We anticipate that the isolated variants of chymotrypsin B can be novel tools available for proteomic studies focusing on detection of post translational modifications.

Engineering substrate specificity of chymotrypsin by directed evolution

247th National Meeting (2014)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.