Investigation into the Chemical Modification of Active Site Residues of an Aldolase Enzyme

It has previously been demonstrated that the biological incorporation and chemical modification of cysteine residues within the active site of the aldolase N-acetylneuraminic acid lyase (NAL) can lead to active chemically modified NAL variants.
This thesis reports the exploitation of this joint chemical/biological method in the incorporation of several unnatural amino acids into the active site of Staphylococcus aureus NAL (SaNAL). This work was performed in an attempt to broaden the substrate specificity of the protein, to allow for the catalysis of the retro-aldol reaction of analogues of N-acetylneuraminic acid (the natural substrate of NAL).
This method was used to incorporate a range of unnatural amino acids into the active site and kinetic parameters of these proteins were assessed with several varied N-acetylneuraminic acid analogues.
The residues F190 and E192 of SaNAL were chosen for chemical modification as they have previously been shown to be important for the catalytic function of the protein.
A coupled enzyme assay was used to assess the kinetic parameters of the chemically modified variants of SaNAL with the analogues of N-acetylneuraminic acid. As a result, it was found that a range of chemically modified amino acids at position 192 of SaNAL would allow for improved specific activity (kcat/KM) of the retro aldol reaction with the substrate DPAH, when compared to wild-type SaNAL with DPAH. An interesting relationship was also observed between the functionality of chemically modified amino acids at position 190 and the specific activity of the resulting protein in the retro-aldol of the substrate N-acetylneuraminic acid.