Major
Chemistry
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Abstract
ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the regulating enzyme of glycogen synthesis in bacteria. The enzyme catalyzes the reaction between glucose 1-phosphate (Glc1P) and ATP to produce ADP-glucose and pyrophosphate. In Agrobacterium tumefaciens it is regulated by fructose 6-phosphate (Fru6P) and pyruvate, and it is inhibited by AMP. The allosteric sites of the enzyme have been extensively studied. Specific residues in these sites have been shown to alter the activity of the enzyme. Previously, Arginine75 when mutated to an alanine was shown to increase the activity of the enzyme. The absence of the positively charged residue caused the enzyme to be in a pre-activated state that can be further activated in the presence of activator, when compared to the wild type. Tyrosine124 when mutated to an alanine showed a pre-activated state as well, but unlike R75A, it was unable to be further activated by the addition of activator. Here we combine these single mutations into a double mutant R75A/Y124A to study the possibility of further increasing the enzymatic activity of ADP-Glc PPase, when compared to the wild type and the individual mutations. In the absence of activator R75A/Y124A was pre-activated and showed a specific activity that matches that of the single mutant R75A in the presence of Fru6P of 220 U/mg. In the presence of both pyruvate and Fru6P the activity of R75A/Y124A mutant was unchanged and resembled the pre-activated enzyme in the absence of activator. In the future, we plan to combine this double mutation with other pre-activated single mutants and see if the enzyme can outperform the enzymatic activity of the established single mutations. This project was funded by a grant from the National Science Foundation (MCB 1616851).
Faculty Mentors & Instructors
Dr. Ballicora, Chemistry & Biochemistry Department Chair; Gaby Martinez-Ramirez; Chemistry & Biochemistry PhD Candidate
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.
Characterizing the Double Mutation R75A/Y124A and its Role in the Allosteric Regulation of ADP-glucose Pyrophosphorylase from Agrobacterium tumefaciens
ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the regulating enzyme of glycogen synthesis in bacteria. The enzyme catalyzes the reaction between glucose 1-phosphate (Glc1P) and ATP to produce ADP-glucose and pyrophosphate. In Agrobacterium tumefaciens it is regulated by fructose 6-phosphate (Fru6P) and pyruvate, and it is inhibited by AMP. The allosteric sites of the enzyme have been extensively studied. Specific residues in these sites have been shown to alter the activity of the enzyme. Previously, Arginine75 when mutated to an alanine was shown to increase the activity of the enzyme. The absence of the positively charged residue caused the enzyme to be in a pre-activated state that can be further activated in the presence of activator, when compared to the wild type. Tyrosine124 when mutated to an alanine showed a pre-activated state as well, but unlike R75A, it was unable to be further activated by the addition of activator. Here we combine these single mutations into a double mutant R75A/Y124A to study the possibility of further increasing the enzymatic activity of ADP-Glc PPase, when compared to the wild type and the individual mutations. In the absence of activator R75A/Y124A was pre-activated and showed a specific activity that matches that of the single mutant R75A in the presence of Fru6P of 220 U/mg. In the presence of both pyruvate and Fru6P the activity of R75A/Y124A mutant was unchanged and resembled the pre-activated enzyme in the absence of activator. In the future, we plan to combine this double mutation with other pre-activated single mutants and see if the enzyme can outperform the enzymatic activity of the established single mutations. This project was funded by a grant from the National Science Foundation (MCB 1616851).