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Download Altering enzyme activities using chemical modification Claire Louise
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P034 Altering enzyme activities using chemical modification Claire Louise Windle, Alan Berry, Arwen R Pearson and Adam S Nelson University of Leeds, Leeds, UK Enzymes are constructed from the twenty proteogenic amino acids. Nature, however has expanded on the available chemistries for example by recruiting cofactors or by inserting non-canonical amino acids. In Nature there are two main ways of inserting these non-canonical amino acids: either by genetic incorporation or by post-translational modification. Major advances in engineering new enzyme activities have been made by site-directed mutagenesis and directed evolution, however these methods are restricted to the use of the twenty proteogenic amino acids. By using chemical modification we have produced a protein containing a non-natural amino acid at high enough yield for detailed kinetic characterisation and crystallographic studies. The catalytically important Lys-165 in the S. aureus N-acetylneuraminic acid lyase (NAL) was converted into a cysteine. Chemical modification was used to convert it into γ-thialysine, via dehydroalanine. The structures of both wild-type and K165-γ-thialysine enzymes have been solved in the presence, and absence, of pyruvate. These studies showed γ-thialysine mimics both the structure of the natural lysine and the binding of the substrate. However kinetic investigation of the K165-γ-thialysine enzyme showed its activity to be lower than that of the wild-type enzyme. A detailed pH-activity profile of the modified enzyme showed that the pH optimum was perturbed from pH 7.4 to 6.8. This study highlights the feasibility of chemical modification for incorporating novel non-canonical amino acids into many enzymes.