Journal Article

Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase

Okita T.W, Woodbury R.L, Greene T.W

1996

Overview

As part of a structure-function analysis of the higher-plant ADP-glucose pyrophosphorylase (AGP), we used a random mutagenesis approach in combination with a novel bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Greene, S.E. Chantler, M.L. Khan, G.F. Barry, I. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509-1513). One mutant of the large subunit M27 was identified by its capacity to only partially complement a mutation in the structural gene for the bacterial AGP (glg C), as determined by its light-staining phenotype when cells were exposed to I2 vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and AGP activity was comparable to those of cells that expressed the wild-type recombinant enzyme. Kinetic analysis indicated that the M27 AGP displays normal Michaelis constant values for the substrates glucose-1-phosphate and ATP but requires 6- to 10-fold greater levels of 3-phosphoglycerate (3-PGA) than the wild-type recombinant enzyme for maximum activation. DNA sequence analysis showed that M27 contains a single point mutation that resulted in the replacement of aspartic acid 413 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PGA. Aspartic acid 413 is adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PGA (K. Ball, J. Preiss [1994] J Biol Chem 269: 24706-24711). The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP.

• Publisher: American Society of Plant Physiologists
• Subject: acide aspartique / acido aspartico / activador enzimatico / activateur d' enzyme / actividad enzimatica / activite enzymatique / adn recombinado / adn recombine / alanina / alanine / Allosteric Regulation / Allosteric Site / almidon / amidon / Amino Acid Sequence / amino acid sequences / Analytical, structural and metabolic biochemistry / Aspartic Acid / Biochemistry and Enzymology / Biological and medical sciences / biosintesis / biosynthese / Biosynthesis / chemical composition / chemistry / composicion quimica / composition chimique / culture media / DNA Primers / enzyme activators / enzyme activity / Enzyme substrates / Enzymes / Enzymes and enzyme inhibitors / enzymic activity / enzymology / Fundamental and applied biological sciences. Psychology / glicogeno / Glucose-1-Phosphate Adenylyltransferase / Glycogen / glycogene / Kinetics / lisina / lysine / Macromolecular Substances / medio de cultivo / Metabolism / milieu de culture / Molecular Sequence Data / mutacion / Mutagenesis / Mutagenesis, Site-Directed / mutation / Nucleotidyltransferases / Nucleotidyltransferases - chemistry / Nucleotidyltransferases - metabolism / phosphotransferases (kinases) / Plant physiology and development / Plants / Point Mutation / recombinant DNA / Recombinant Proteins / Recombinant Proteins - chemistry / Recombinant Proteins - metabolism / Sequence Homology, Amino Acid / site-directed mutagenesis / Solanum tuberosum / Solanum tuberosum - enzymology / Spinacia oleracea / Spinacia oleracea - enzymology / starch / Starches / strains / transferasas / transferase / Transferases / Tubers