Download Altering enzyme activities using chemical modification Claire Louise

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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.