Date of Award
Master of Science (MS)
UDP-Glucose Pyrophosphorylase (UDP-Glc PPase) is a key enzyme of the carbohydrate metabolic pathway widely used among prokaryotes and eukaryotes. In plants, UDP-Glc PPase is necessary for sucrose synthesis while mammals utilize this enzyme for the production of glycogen. A bacterium such as Escherichia coli uses UDP-Glc PPase for biosynthesis of the lipopolysscharide core which forms a cell wall.
This study focused on UDP-Glc PPase from Escherichia coli which is encoded by both galU and galF genes. The first part of this project investigated amino acids that could play an important role in the function of UDP Glc PPase (GalU). Based on the UDP-Glucose Pyrophosphorylase (GalU) model with Corynebacterium glutamicum's UDP-Glucose and magnesium ion, we hypothesized that Glutamic acid- 201, Glutamine-109, Lysine- 202, Arginine-21, Lysine-31, and Aspartic acid 265 are residues that play a critical role in UDP-Glc PPase (GalU) either in catalysis or binding of substrates.
The second part of this project focused on finding whether UDP-Glc PPase from the galF gene is catalytic or not. Also, the alignment of eukaryotic galU and galF amino acid sequences showed that Threonine-20 and Arginine-21 side chains are missing in GalF. Therefore, we hypothesized that by mutating these two residues in the galF gene, GalF activity will be resurrected (if wild type is inactive) or mutations will increase the enzyme's activity (if wild type is active).
The catalytic characterization of GalU revealed a decrease in UDP-Glucose Pyrophosphorylase activity of all mutants. Out of all mutated residues, Arginine-21 was the most critical catalytic amino acid since the mutation showed the highest drop in activity compared to wild type. Glutamine-109 was important for the binding with Uracil, demonstrating its specificity for this type of enzyme. Lysine-202 and Glutamic Acid-201 had recognition for the sugar, UDP-glucose, but they are not important for the enzyme's specificity. Aspartic Acid-201 indirectly participated in the pyrophosphate binding.
Magnesium ions were critical for UDP-Glc PPase's activity. Furthermore, approximately 2 mM was the minimal concentration of magnesium necessary for the enzyme's maximum activity.
It has been reported that the galF gene does not encode an active UDP-Glc PPase, however, our study revealed that GalF is an active enzyme. Yet, the protein's specific activity was 100-fold lower compared to GalU. This result suggests that the ancestry of GalF is a catalytic subunit and that it became reduced in activity as it evolved.
The saturation curve revealed that GalF needs magnesium ions in order to be active. The comparison of binding affinities between GalF and GalU showed that GalF requires a higher concentration of magnesium in order for its maximal activity to be detected. The two mutations performed on the galF gene did not affect GalF's activity and they did not have an effect on the binding affinity of UDP-Glc or PPi.
Orlof, Agnieszka Maria, "Structure-Function Relationship Studies of the UDP-Glucose Pyrophosphorylase From Escherichia Coli" (2010). Master's Theses. 558.
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Copyright © 2010 Agnieszka Maria Orlof