SummaryAsp142in the homotetrameric ADP‐glucose pyrophosphorylase (ADP‐Glc PPase) enzyme fromEscherichia coliwas demonstrated to be involved in catalysis of this enzyme [Frueauf, J.B., Ballicora, M.A. and Preiss J. (2001)J. Biol. Chem., 276, 46319–46325]. The residue is highly conserved throughout the family of ADP‐Glc PPases, as well as throughout the super‐family of sugar–nucleotide pyrophosphorylases. In the heterotetrameric ADP‐Glc PPase from potato (Solanum tuberosumL.) tuber, the homologous residue is present in both the small (Asp145) and the large (Asp160) subunits. It has been proposed that the small subunit of plant ADP‐Glc PPases is catalytic, while the large subunit is modulatory; however, no catalytic residues have been identified. To investigate the function of these conserved Asp residues in the ADP‐Glc PPase from potato tuber, we used site‐directed mutagenesis to introduce either an Asn or a Glu. Kinetic analysis in the direction of synthesis or pyrophosphorolysis of ADP‐Glc showed a significant decrease (more than four orders of magnitude) in the specific activity of the SD145NLwt, SD145NLD160N, and SD145NLD160Emutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160Emutants. By contrast, mutation of the large subunit alone did not affect the specific activity but did alter the apparent affinity for the activator 3‐phosphoglycerate, showing two types of apparent roles for this residue in the different subunits. These results show that mutation of Asp160of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145in the small subunit is necessary for enzyme catalysis.