Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.643350
Title: The purification and characterisation of a mutant form of pyruvate kinase from Saccharomyces cerevisiae produced by site-directed mutagenesis
Author: Collins, Richard A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1994
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Abstract:
A variant form of the glycolytic enzyme pyruvate kinase (E.C.2.7.1.40) in which the serine residue at position 384 of the polypeptide chain has been mutated to proline has been overexpressed in the yeast Saccharomyces cerevisiae. The mutant protein has been purified to homogeneity and has been characterised by a number of physical, kinetic and chemical techniques. The properties of the mutant enzyme have been compared to those of the native wild type enzyme overexpressed from the same organism. The wild type enzyme is activated by the allosteric effector fructose-1,6-bisphosphate. The mutant enzyme was found to be dependent upon the presence of this effector for catalytic activity and was inactive in its absence. The mutant enzyme became 50% activated at 1.9 ± 0.1mM Fru-1,6-P2. In the absence of the effector the kcat of the mutant enzyme was reduced by a factor of 250. The fully activated mutant enzyme had a kcat 68% of that of the wild type enzyme. The mutation introduced into the enzyme was proposed to be at a site critical for the transfer of the allosteric effect across the enzyme upon effector binding. This hypothesis is demonstrated to be correct, and can be extended to invoke additional conformational changes at the native site, as the mutant enzyme displayed different kinetic properties to the wild type enzyme in the presence of the allosteric effector fructose-1,6-bisphosphate. The S0.5PEP and S0.5ADP of the wild type enzyme were 0.15mM and 0.39mM respectively. In the mutant enzyme, these kinetic parameters increased to 0.69 and 0.94mM respectively. The cooperativity between binding sites also increased significantly in the mutant enzyme. The mutant also displayed altered monovalent and divalent cation specificities and an altered pH profile in the presence and absence of saturating concentrations of effector. In the presence of effector, the mutant enzyme had a narrower pH profile than the wild type enzyme although both enzymes displayed maximal activity at pH 6.5.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.643350  DOI: Not available
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