Engineering enzymes for novel catalysis

Abstract

Part one of this thesis outlines an attempted incorporation of a new functionality into the active site of {u03B1}-chymotrypsin through post-translational chemical modification. Model compound studies, whole protein mass spectrometry, UV spectroscopy studies, as well as cycloaddition reactions have shown that the inhibited {u03B1}-chymotrypsin undergoes an elimination reaction thereby converting the active site serine to a dehydroalanine rather than a substitution product. These results clearly demonstrate that other reactive non-proteinogenic residues could be introduced into the active site of a-chymotrypsin via a cycloaddition or halogenation reaction with the generated dehydroalanine side chain. It is likely that such incorporation may meet with success thereby giving the newly generated enzyme novel catalytic activity. The second part of the thesis explored the use of cell-free S30 extract from E. coli for novel catalysis. Investigations conducted previously showed that S30 extract converted (S)-fluoroleucine hydrazide to (S)-fluoroleucine with subsequent incorporation into ubiquitin. Work presented in this thesis has established that the S30 extract does not only cleave the hydrazide and methyl ester of leucine but also those of phenylalanine and valine. Through stereospecificity experiments and inhibition studies, it was established that the observed hydrazide cleavage was due to an enzymatic action with the enzymes likely to be metalloproteases. A further literature search showed that the S30 extract used in many protein syntheses contains a lot of enzymes, most of whose activity are not inhibited by PMSF, added to the cell-free S30 extract during preparation. Several experiments were conducted using HPLC techniques to establish that the S30 extract has a broad specificity towards the cleavage of a variety of amino acid hydrazides. Further investigations on the scope and limitations of the S30 extract enzymes towards the cleavage of other protecting groups indicated that they are not limited to cleaving only hydrazides but also possess catalytic capability towards the removal of frequently used amino acid protecting groups such as N-acetyl, N-trifluoroacetyl, benzyl ester, N-benzoyl, t-butyl amide and adamantanylamide. It was also established that the S30 extract can remove protecting groups from a fully protected amino acid in a single step. Through the techniques of whole protein mass spectrometry and SDS-PAGE analysis, it was shown that the removal of various investigated protecting groups by S30 proceeded efficiently, with the resultant free amino acids subsequently incorporated into His{u2086}-PpiB in a one-pot protein synthesis reaction. This shows that the steps involved in the preparation of chemically manipulated amino acids can be reduced by adding the protected, chemically manipulated (non-proteinogenic) amino acid to the cell-free S30 extract for protein synthesis without any deprotection. This was illustrated with some preliminary results presented in the last chapter of this thesis with the successful incorporation of a dehydroleucine into His{u2086}-PpiB through the addition of the protected derivative (dehydroleucine hydrazide) to the protein synthesis mixture instead of leucine.