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.
Mahaffey, Angela, Saleh Aiyash, Miguel Ballicora, Ligin Solamen. "Investigating the Electrostatic Role of a Critical Arginine for the Catalysis of E. Coli ADP-Glucose Pyrophosphorylase." Biophysical Journal, Volume 104, Issue 2, Supplement 1, January 29, 2013, http://dx.doi.org/10.1016/j.bpj.2012.11.3087.
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