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Title: Protein engineering studies on phosphoglycerate mutase from Saccharomyces cerevisiae
Author: Walter, Rebecca
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1999
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The glycolytic enzyme phosphoglycerate mutase from Saccharomyces cerevisiae catalyzes the interconversion of 2- and 3-phosphoglycerate via a Ping-Pong mechanism involving a phosphorylated histidine residue. It also shows a low level of phosphatase activity which is thought to be a result of the non-productive transfer of a phospho group to water. The two C-terminal residues are both lysines, and it has been suggested that they are involved in catalysis. Three mutants in which one or both lysines are replaced by glycine have been characterized (K246G, K245G and K245G/K246G). The K246G mutant has kinetic properties almost identical to the wild type. When the lysine 245 residue is replaced, there are some differences in the kinetic parameters of the resulting mutants which suggest changes in the binding of the substrate and/or release of the product, but it is unlikely that either lysine is involved in electrostatic interactions crucial to the catalytic mechanism. Phosphoglycerate mutase also catalyzes a 2,3-bisphophoglycerate synthase activity although at a much lower level than the mutase activity, and the yeast enzyme is closely related to the bisophosphoglycerate synthase enzyme present in red blood cells. This enzyme catalyzes the same three reactions as the yeast mutase, but the synthase activity is much higher in the erythrocyte enzyme. Comparison of the amino acid sequences of the two proteins show two significant differences in the active site region: residues 11 and 60 (S. cerevisiae numbering) are a serine and an alanine respectively in the yeast mutase, and a glycine and a serine in the erythrocyte synthase. Characterization of yeast mutase mutants with residues 11 and 60 replaced by the corresponding synthase residues (S11A and A60S) showed differences in the catalytic parameters and decreased mutase activity. However, circular dichroism shows that this could be due to structural differences rather than a change in functional groups in the active site. Another active site residue implicated in the catalytic mechanism is glutamate 86. When this residue is replaced with a glutamine, there is a very significant decrease in mutase activity, and the KM values for each of the substrates are increased significantly. It has been suggested that this residue is involved in a catalytic triad in the related enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. These results support a similar role for the residue in the mutase activity of the S. cerevisiae phosphoglycerate mutase.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available