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Title: Studies on the mechanism of porphobilinogen deaminase using site directed mutagenesis
Author: Woodcock, Sarah Catherine
ISNI:       0000 0001 3572 1498
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 1992
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Site directed mutagenesis of the E.coli porphobilinogen deaminase gene was performed in order to investigate the role of twelve highly conserved arginine residues within the protein molecule. A series of mutants of the hemC gene were generated, in which arginine was replaced by histidine, using the Eckstein method of mutagenesis, and these were identified by DNA sequencing. The mutant genes were cloned into the expression vector, pUC18. The mutant proteins were purified to homogeneity and were all found to have a molecular weight of 35,000Da, the same as the wild-type enzyme. The mutant proteins were categorised and the ability of each protein to produce enzyme-intermediate complexes was studied. The role of each mutated arginine residue within the porphobilinogen deaminase molecule was suggested in the light of the mutagenesis results and the three dimensional X-ray structure. Only two of the twelve arginine residues, arginine-131 and arginine-132 were found to be essential for the binding of the dipyrromethane cofactor. Arginine-11 and arginine-155 were found to be required for activity although both still have the cofactor. Arginines-149 and -176 were found to be involved in the binding and/or movement of the growing tetrapyrrole chain through the active site cleft. Arginine-232 was found to be located away from the active site and appears to have an important structural role. In addition aspartate-84, which from the X-ray structure of the wild-type enzyme was seen to be located between the two pyrrole nitrogens of the cofactor, was found to have a catalytic role. Three mutants were generated in which aspartate was replaced by alanine, asparagine and glutamate. The alanine and asparagine mutants were found to be devoid of activity, although they contained the cofactor. However the glutamate mutant, D84E, had a greatly reduced specific activity and was shown to accumulate enzyme intermediate complexes. It is suggested that aspartate-84 plays an important role in the acid catalytic mechanism.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry