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Doctor of Philosophy (PhD)




Carbohydrates have been most notable as energy sources for mammals, bacteria (glycogen) and plants (starch) – and in many other species. As such the biosynthesis of carbohydrates is essential to the sustainability of many forms of life, on earth. Adenosine-‘5-diphosphate glucose pyrophosphate (ADP-Glc pyrophosphorylase; ADP-Glc PPase) is the allosterically controlled “first committed step” in both the biosynthetic pathways of starch (~25% amylose and ~75% amylopectin, in plants and algae) and glycogen (in bacteria), preceding the starch/glycogen synthase reaction. By catalyzing the following reaction, ATP + α –D-Glc-1P ADP-Glc + PPi , ADP-Glc PPase functions as the primary enzyme in the reaction that provides the glycosyl precursor for the elongation of α-1,4-polyglucans. ADP-Glc PPase is a tetrameric allosterically regulated enzyme. The structure of this enzyme is homotetrameric in enteric bacteria (Escherichia coli) – α4, and heterotetrameric in plants and other photosynthetic eukaryotes – consisting of two small subunits (50-52 kDa) and two large subunits (~60kDa): (α2β2). The plant ADP-Glc PPases are allosterically regulated: mainly by 3-phosphoglycerate (3PGA) as an activator and inhibited in the presence of inorganic phosphate (Pi). There is an allosteric disparity in the bacterial enzyme’s counterpart. For instance, the E. coli ADP-Glc PPases enzyme is activated by fructose-1,6-bisphosphate (FBP) and inhibited by adenosine-‘5-monophosphate (AMP). All ADP-Glc PPases, to date, are noted to have a divalent metal ion cofactor (Mg+2).

Comparative modeling of the E. coli ADP-Glc PPase with ATP bound in the active site predicted critical interactions for Lys42. In the model, this residue interacts with the catalytic Asp142 and the β-phosphate of the ATP substrate, which comprises the reaction “leaving group”. Lys42 is highly conserved in the ADP-Glc PPases known to be catalytic, but absent in plant subunits that are catalytically deficient. It is also conserved in other homologues of the sugar-nucleotide pyrophosphorylase superfamily. To investigate the role of Lys42 in E. coli ADP-Glc PPase, we performed site-directed mutagenesis. As a result, we observed a markedly decreased kcat (>3 orders of magnitude lower than the wild type (WT)).

We analyzed another conserved residue that interacts with the phosphates of ATP (Arg32). In mutating the Arg32 residue, we observed the S0.5 of the ATP substrate for the mutants was only two to three times higher than that of the WT. But more significant was the marked decrease in specific activity (and kcat) for the Arg32 mutants (1 -3 orders of magnitude). Our results indicate that the interaction of Arg32 guanidinium moeity and structural length of the Arg32 side chain is critical for overall catalysis. Modeling of the E. coli enzyme WT and Arg32 mutants suggest that two nitrogen atoms of the Arg32 guanidinium side chain may interact with the γ-phosphate of the ATP substrate making the PPi product a more stable leaving group. These results show that both residues in the E. coli ADP-Glc PPase are catalytic (Lys42), important for orientation and positioning of the ATP substrate (Arg32) and overall essential for the production of ADP-glucose.

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

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