Studies on recombinant ubiquitous and erythroid human porphobilinogen deaminase and mutational analysis of E. Coli porphobilinogen deaminase

Abstract

Porphobilinogen deaminase, the third enzyme of the haem biosynthesis pathway catalyses the stepwise tetramerisation of porphobilinogen to form the unstable hydroxymethylbilane, preuroporphyrinogen. In humans, non-erythroid (ubiquitous) and erythroid of porphobilinogen deaminase are both specified by a single 10 kilobase gene comprising 15 exons.

Experiments in this thesis are described in which the cDNA specifying human porphobilinogen deaminase was cloned so that both ubiquitous and erythroid forms could be over-expressed in E. coli BL21(DE3).  After purification to homogeneity, both isoenzymes were characterized by physical and chemical techniques, including mass spectrometry, amino-terminal sequencing and kinetic studies.  Crystals of ubiquitous and erythroid porphobilinogen deaminases were produced using the hanging drop vapour diffusion method, and X-ray crystallographic data were collected for the erythroid isoform to 3.0A resolution.  A three-dimensional X-ray model was built by molecular replacement using the known human ubiquitous porphobilinogen deaminase arginine 167 glutamine mutant structure.

Experiments were also carried out on two mutants of E. coli porphobilinogen deaminase, Gly 57 Ala and Gly 264 Cys.  Gly 57 is located on a mobile active site strand in domain 1 and Gly 264 is a surface residue of domain 3 close to Cys 134  in domain 2. It was hoped that investigations on these two mutants would provide information about conformational changes in the enzyme during the catalytic cycle.

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