ADP-glucose pyrophosphorylase from potato tuber: site-directed mutagenesis of homologous aspartic acid residues in the small and large subunits.
Asp142 in the homotetrameric ADP-glucose pyrophosphorylase (ADP-Glc PPase) enzyme from Escherichia coli was 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 tuberosum L.) 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 SD145NLD160E mutants, while the effect was smaller (approximately two orders of magnitude) with the SD145ELwt, SD145ELD160N, and SD145ELD160E mutants. 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 Asp160 of the large subunit does not affect catalysis, thus the large subunit is not catalytic, and that the negative charge of Asp145 in the small subunit is necessary for enzyme catalysis.
Frueauf
Jeremiah B
JB
Department of Biochemistry and Molecular Biology, Michigan State University, Biochemistry Building, East Lansing, MI 48824, USA.
Ballicora
Miguel A
MA
Preiss
Jack
J
D001224:Aspartic Acid / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D001224:Aspartic Acid; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase