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Title: Investigation of the structure and function of 5-aminolaevulinic acid dehydratases
Author: Senior, Natalie Marianne
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1997
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5-Aminolaevulinic acid dehydratase (ALAD) catalyses an inaugural step in the tetrapyrrole biosynthetic pathway, the Knorr type condensation of two molecules of 5-aminolaevulinic acid (ALA) to form the monopyrrole, porphobilinogen. The cloning and overexpression of ALAD from Saccharomyces cerevisiae (yeast) is described with the purification to homogeneity of this enzyme and the ALADs from Pisum sativum (pea) and Escherichia coli (E. coli). Comparative studies on these three ALADs revealed differences between them. In particular, the enzyme from pea is magnesium dependent whereas that from yeast is zinc dependent but able to bind magnesium and E. coli ALAD is zinc dependent but magnesium stimulated. All three enzymes are homo-octameric and protein modifying reagents and pH dependent kinetics revealed that the enzymes have important lysine, cysteine and histidine residues. Lysines are probably involved in the formation of a Schiff base with the carbonyl group on ALA which could be trapped with sodium borohydride. Metals were required for catalysis but not for the formation of the Schiff base. Circular dichroism revealed that the enzymes have similar secondary structure and this is remarkably stable to denaturants. Conformational changes on metal loss or substrate binding are minimal although chelating agents and denaturants appear to facilitate the loosening of the ALAD octameric structure. The enzymes are also heat sensitive, showing two stages of denaturation. Studies with inhibitors revealed the different steric constraints on active site binding between the enzymes and showed that source selective enzyme inhibition could be achieved. In particular, the inhibitor succinylacetone has Ki values that vary by three orders of magnitude. In addition, the crystallisation, heavy metal deriv isation and site directed mutagenesis of yeast ALAD is reported with the progress towards an X-ray derived three dimensional structure for the enzyme.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available