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Structural and mechanistic studies on E.coli porphobilinogen deaminase and mutant variants

Awan, Sarah Jabeen; (1996) Structural and mechanistic studies on E.coli porphobilinogen deaminase and mutant variants. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Structural and mechanistic studies have been carried out on porphobilinogen deaminase, the third enzyme in the tetrapyrrole biosynthesis pathway. The enzyme catalyses the tetrapolymerisation of porphobilinogen to yield the hydroxymethylbilane product, preuroporphyrinogen. The holoenzyme possesses a novel dipyrromethane cofactor (itself derived from two molecules of porphobilinogen) to which the substrate molecules are bound via the enzyme-intermediate complexes ES, ES2, ES3 and ES4 Two aspects of the enzyme have been studied (i) the role of the invariant and catalytically important residue aspartate-84 and (ii) the conversion of apoenzyme (enzyme lacking the cofactor) to holoenzyme. Three mutants of aspartate-84 were purified and characterised (D84A, D84E and D84N). The D84E mutant was found to exist as free enzyme and also stable enzyme-intermediate complexes. The D84E free enzyme form (E) was crystallised, providing a molecular insight into its catalytic incapacity. Furthermore, the increased stability of the enzyme-intermediate complexes led to the crystallisation of the ES2 complex, and thus the elucidation of the enzyme in the midst of its catalytic cycle. The D84A and D84N mutants were found to be completely inactive yet existed as ES2 complexes. An explanation for this phenomenon came from studies on the reconstitution of holoenzyme from apoenzyme. The apoenzyme was functionally and structurally characterised. Circular dichroism studies revealed considerable secondary structure for the apo-protein, although the apparent susceptibility to modification and denaturation was more indicative of an open conformation. It is proposed that the domains may be tightly packed, but flexible about interdomain hinges due to the absence of the cofactor. The transformation from apoenzyme to holoenzyme was also thoroughly investigated. It was found that whilst the apoenzyme was able to bind porphobilinogen to reconstitute the holoenzyme, probably using the same machinery as employed by holoenzyme for catalysis, the preferred substrate was, in fact, preuroporphyrinogen. This finding gives rise to a new theory on cofactor assembly, and explains the existence of the ES2 complexes for the catalytically inactive D84A and D84N mutants.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structural and mechanistic studies on E.coli porphobilinogen deaminase and mutant variants
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Health and environmental sciences; E.coli
URI: https://discovery.ucl.ac.uk/id/eprint/10100570
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