Rational design of artificial enzymes

Muroni, Maurizio 2011. Rational design of artificial enzymes. PhD Thesis, Cardiff University.

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Abstract

Despite the endeavours of many decades, the design of artificial enzymes remains challenging. The work presented here investigates two known molecules as scaffolds for the design of artificial enzymes an 18 amino acids a helical peptide with two disulfide bridges - 'Apoxaldie' able to catalyse the decarboxylation of oxaloacetate, and the 86-amino acid colicin E9 immunity protein (Im9) with four a helices. Apoxaldie was modified such that the active site lysines were substituted by 2,4-diaminobutyric acid in order to increase the proximity of the enzyme active site to the chiral environment of the a helix. The designed peptide ('Apoxaldie-Dab') was synthesized with two different strategies and the correct formation of disulfide bonds was achieved. However, Apoxaldie-Dab did not show the expected activity for the decarboxylation of oxaloacetate. Circular dichroism studies showed a 30% loss of a helicity upon introduction of 2,4- diaminobutyric acid into Apoxaldie which can explain the decrease in activity. In the case of Im9, two series of mutants, constructed around histidine 10 and asparagine 78 respectively, were designed to introduce histidine-based active sites into hydrophobic clefts. Site directed mutagenesis, gene expression and variant protein purification were carried out for ten variant mutants together with the wild type. The secondary structure and thermal stability of each protein were studied and catalytic activities were examined by monitoring the hydrolysis of /-nitrophenol acetate via ultraviolet-visible spectroscopy. The Im9 variant Tm9-W74A/N78H' demonstrated three times more activity compared to Im9, indicating the modification of IM9 to possess a histidine based active site increased activity as hypothesized through its rational design. The initial work of applying directed evolution on Tm9-W74A/N78H' was accomplished by constructing a phage display library. A transition state analogue was synthesised to test screening the expressed library. This method can be further developed to assist the design of Im9-based artificial enzymes.

Item Type:	Thesis (PhD)
Status:	Unpublished
Schools:	Chemistry
Subjects:	Q Science > QD Chemistry
ISBN:	9781303218989
Date of First Compliant Deposit:	30 March 2016
Last Modified:	19 Mar 2016 23:31
URI:	https://orca.cardiff.ac.uk/id/eprint/55043

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