ADP-Glucose pyrophosphorylase from potato tubers. Site-directed mutagenesis studies of the regulatory sites.
Several lysines (Lys) were determined to be involved in the regulation of the ADP-glucose (Glc) pyrophosphorylase from spinach leaf and the cyanobacterium Anabaena sp. PCC 7120 (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711; Y. Charng, A.A. Iglesias, J. Preiss [1994] J Biol Chem 269: 24107-24113). Site-directed mutagenesis was used to investigate the relative roles of the conserved Lys in the heterotetrameric enzyme from potato (Solanum tuberosum L.) tubers. Mutations to alanine of Lys-404 and Lys-441 on the small subunit decreased the apparent affinity for the activator, 3-phosphoglycerate, by 3090- and 54-fold, respectively. The apparent affinity for the inhibitor, phosphate, decreased greater than 400-fold. Mutation of Lys-441 to glutamic acid showed even larger effects. When Lys-417 and Lys-455 on the large subunit were mutated to alanine, the phosphate inhibition was not altered and the apparent affinity for the activator decreased only 9- and 3-fold, respectively. Mutations of these residues to glutamic acid only decreased the affinity for the activator 12- and 5-fold, respectively. No significant changes were observed on other kinetic constants for the substrates ADP-Glc, pyrophosphate, and Mg2+. These data indicate that Lys-404 and Lys-441 on the small subunit are more important for the regulation of the ADP-Glc pyrophosphorylase than their homologous residues in the large subunit.
Ballicora
M A
MA
Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Fu
Y
Y
Nesbitt
N M
NM
Preiss
J
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D015870:Gene Expression; D051599:Glucose-1-Phosphate Adenylyltransferase; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010710:Phosphates / Q000494:pharmacology; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics*
D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D010710:Phosphates; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase