ADP-Glc pyrophosphorylase (PPase), a key regulatory enzyme in the biosynthetic pathway of starch and bacterial glycogen, catalyzes the synthesis of ADP-Glc from Glc-1-P and ATP. A homology model of the three-dimensional structure of the Escherichia coli enzyme complexed with ADP-Glc has been generated to study the substrate-binding site in detail. A set of amino acids in the model has been identified to be in close proximity to the glucose moiety of the ADP-Glc ligand. ) was studied by site-directed mutagenesis through the characterization of the kinetic properties and thermal stability of the designed mutants. All purified alanine mutants had 1 or 2 orders of magnitude lower apparent affinity for Glc-1-P compared with the wild type, indicating that the selected set of amino acids plays an important role in their interaction with the substrate. These amino acids, which are conserved within the ADP-Glc PPase family, were replaced with other residues to investigate the effect of size, hydrophobicity, polarity, aromaticity, or charge on the affinity for Glc-1-P. In this study, the architecture of the Glc-1-P-binding site is characterized. The model overlaps with the Glc-1-P site of other PPases such as Pseudomonas aeruginosa dTDP-Glc PPase and Salmonella typhi CDPGlc PPase. Therefore, the data reported here may have implications for other members of the nucleotide-diphosphoglucose PPase family.The biosynthetic pathways of starch and bacterial glycogen are very similar (1). The initial and key regulatory step is the formation of the glucosyl donor molecule ADP-Glc from ATP and Glc-1-P via a reaction catalyzed by ADP-glucose pyrophosphorylase (PPase 4 ; glucose-1-phosphate adenylyltransferase, EC 2.7.7.27), with the requirement of a divalent cation (Mg 2ϩ ): ATP ϩ Glc-1-P 4 Mg 2ϩ 3 ADP-Glc ϩ PP i . Most ADP-Glc PPases are allosterically regulated by small effector molecules. Although these vary according to the source, they are all intermediates of the principal carbon assimilation pathway in the respective cell (2-6). Thus, bacterial glycogen and plant starch syntheses are not modulated only by the availability of ATP but also by the accumulation of key metabolites that represent the carbon and energy balance within the cell. For instance, the enzymes from heterotrophic bacteria such as Escherichia coli are regulated by intermediates of the glycolytic pathway, with Fru-1,6-P 2 as the main activator and AMP as the main inhibitor. On another hand, the ADP-Glc PPases from cells performing oxygenic photosynthesis and assimilating atmospheric CO 2 through the reductive pentose phosphate pathway or the Calvin cycle (specifically cyanobacteria, green algae, and photosynthetic tissues from higher plants) are activated by 3-phosphoglycerate and inhibited by P i (6).Except for some Bacillus species (5-7), prokaryotic ADP-Glc PPases are homotetramers, with the monomer being ϳ50 kDa (2,5,8,9). Characterized ADP-Glc PPases from higher plants are heterotetramers of two different but homologous subunits (2-6), the "small" or catal...