Altering enzyme activities using chemical modification

In Nature there are twenty proteogenic amino acid ‘building blocks’, from which proteins and enzymes are constructed. These proteogenic amino acids confer activity to enzymes; however there are many instances where the chemistries provided by these ‘building blocks’ are expanded upon. Nature recruits an array of cofactors, post translational modifications and post translationally generated cofactors, all which help to provide function or activity. Until recently the protein engineer was restricted to the use of the twenty proteogenic amino acids, and so access to this increased chemical diversity was highly challenging.
In this thesis, chemical modification has been used to insert a variety of non canonical amino acids (ncAAs) throughout the active site of the enzyme N acetylneuraminic acid lyase (NAL). This modification method incorporates ncAAs site specifically into a protein, via a dehydroalanine intermediate and conjugate addition with a thiol compound. Initial work using this method replaced the catalytic lysine at position 165 with the non canonical analogue γ thialysine. It was possible to obtain homogenously modified protein in high yields for detailed kinetic and X ray crystallographic studies, and therefore possible to elucidate the catalytic and structural consequences of this modification.
The work to replace Lys165 with a non canonical analogue provided a starting point to expand the incorporation of ncAAs into NAL. A total of thirteen different non canonical side chains were incorporated, individually, at thirteen different positions within the active site of NAL. These modified enzymes were then screened for activity with ten different substrates to determine the effects of ncAA incorporation. It was seen that the ncAAs were well tolerated by the enzyme, as active modified enzymes were produced. By incorporating ncAAs it was possible to alter the substrate specificity of the enzyme. The modified enzyme F190Dpc, containing a dihydroxypropyl cysteine side chain, was found to have an increased activity with an altered substrate, erythrose. This activity was higher than the wild type enzyme with both the altered substrate and the wild type substrate, and the non canonical Dpc side chain outperformed any of the proteogenic amino acids when inserted at the same position in the protein, for the substrate erythrose.
This research begins to explore the possibilities of what may be achieved by use of ncAAs. Facile incorporation of ncAAs will allow protein engineers to take inspiration from Nature and expand the chemistries provided by the proteogenic amino acids, hopefully to engineer novel activities or catalysis.