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Biophysical Journal







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ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the regulatory enzyme of the pathway for starch synthesis in plants and glycogen in mammals and enteric bacteria. It exists as a 200 kDa homotetramer (α4) in enteric bacteria, and as a heterotetramer (α2β2) in plants. In both in vivo and in vitro the substrates (Glucose 1-Phosphate; Glc-1P and Adenosine 5'-Triphosphate; ATP) are converted into a glucose donor ADP-Glucose and a pyrophosphate (PPi) via the ADP-Glc PPase enzyme. It has been noted that some residues are conserved in homotetrameric bacterial ADP-Glc PPases, but are not in some plant forms. One of them is Arginine-32 (R32) in the Escherichia coli ADP-Glc PPase. To explore the overall role of this residue and evaluate the structural and electrostatic importance of the Arginine's guanidinium group, we replaced it with Lysine (K, -amino group), Alanine (A, - methyl group), Cysteine (C, -sulfide group), Glutamic (E, - carboxylate group), Glutamine (Q, -amido group) and Leucine (L, -hydrophobic side chain) via site directed mutagenesis. We over-expressed the enzymes, purified them to homogeneity, and measured their kinetic properties. The Specific Activity (U/mg) for the mutants were as follows: WT (90.56), R32A (1.65), R32C (0.57), R32E (0.04), R32K (5.81), R32L (0.65) and R32Q (1.37). Currently, the properties of the R32H (Histidine, -imidizoleum ring) mutant are being investigated. Our results clearly indicate that this guanidinium group of the Arginine-32 residue is critical for catalysis. Modeling of the E. coli enzyme suggests that the two (2) nitrogen atoms of the guanidinium group may interact with the β and γ phosphates of the ATP, helping in the positioning of the substrates, via electrostatic interactions, and making the PPi product a more stable leaving group.


Author Posting © 2013 Biophysical Society. This article is posted here by permission of Elsevier for personal use, not for redistribution. The article was published in the Biophysical Journal, Volume 104, Issue 2, Supplement 1, January 29, 2013,

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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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