Investigations into the biosynthesis of uroporphyrinogen III from porphobilinogen

Leadbetter, Robert Elwyn (1997) Investigations into the biosynthesis of uroporphyrinogen III from porphobilinogen. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

The tetrapolymerisation reaction performed by porphobilinogen deaminase and the subsequent transformation of the linear 1-hydroymethylbilane product, preuroporphyrinogen, into the tetracyclic intermediate uroporphyrinogen III were investigated.

The porphobilinogen deaminase enzyme has been extensively characterised with detailed site directed mutagenesis and X-ray crystallographic studies resulting in a good general understanding of its mechanism of action. In this study several pyrrolic analogues were chemically synthesised by Jack Cheung and their effects on the interaction between porphobilinogen deaminase and its monopyrrolic substrate, porphobilinogen, were determined. Whereas most of these analogues were shown to act as competitive inhibitors, the α,α'-diaminomethyl, α,α'-dihydroxymethyl and α-hydroxymethyl-α'-methyl porphobilinogen analogues were all shown to interact covalently with the enzyme, each forming a stable but reversible enzyme-intermediate complex which blocked the polymerisation reaction. The importance of the α and β substituents are discussed with respect to their role in substrate recognition.

Unlike porphobilinogen deaminase, little is known about the mechanism of action of uroporphyrinogen III synthase. Due to the inherent instability of both the enzyme and its substrate, no site-directed mutagenesis studies have been reported other than the effects of natural mutations in the human enzyme that cause congenital erythropoietic porphyria. For the purpose of the study several mutants of uroporphyrinogen III synthase from Escherichia coli and Bacillus subtilis were generated by site-directed mutagenesis and expressed in Escherichia coli. Three highly conserved residues (tyrosine, serine and threonine) and two highly conserved residues (arginine and glutamic acid) were targeted for detailed investigations. The effects of these mutations on catalysis and the likely roles of each substituted residue are discussed. Initial attempts to crystallise the uroporphyrinogen III synthase enzyme in the presence of a spirolactam inhibitor are currently in progress.

This record has no associated files available for download.