Aspartic Acid 413 1 s lmportant for the Normal Allosteric

As part of a structure-function analysis of the higher-plant ADPglucose pyrophosphorylase (ACP), we used a random mutagenesis approach in combination with a nove1 bacterial complementation system to isolate over 100 mutants that were defective in glycogen production (T.W. Creene, S.E. Chantler, M.L. Khan, C.F. Barry, j. Preiss, T.W. Okita [1996] Proc Natl Acad Sci USA 93: 1509-1513). One mutant of the large subunit M27 was identified by i ts capacity to only partially complement a mutation in the structural gene for the bacterial ACP (glg C), as determined by i ts light-staining phenotype when cells were exposed to I , vapors. Enzyme-linked immunosorbent assay and enzymatic pyrophosphorylysis assays of M27 cell extracts showed that the level of expression and ACP activity was comparable to those of cells that expressed the wildtype recombinant enzyme. Kinetic analysis indicated that the M27 ACP displays normal Michaelis constant values for the substrates glucose-1 -phosphate and ATP but requires 6to 1 O-fold greater levels of 3-phosphoglycerate (3-PCA) 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 41 3 to alanine. Substitution of a lysine residue at this site almost completely abolished activation by 3-PCA. Aspartic acid 413 i s adjacent to a lysine residue that was previously identified by chemical modification studies to be important in the binding of 3-PCA (K. Ball, j. Preiss 119941 ] Biol Chem 269: 24706-2471 1 ) . l h e kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higherplant ACP.