Abstract
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, J. 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 l3 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.
Full Text
The Full Text of this article is available as a PDF (619.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ball K., Preiss J. Allosteric sites of the large subunit of the spinach leaf ADPglucose pyrophosphorylase. J Biol Chem. 1994 Oct 7;269(40):24706–24711. [PubMed] [Google Scholar]
- Ballicora M. A., Laughlin M. J., Fu Y., Okita T. W., Barry G. F., Preiss J. Adenosine 5'-diphosphate-glucose pyrophosphorylase from potato tuber. Significance of the N terminus of the small subunit for catalytic properties and heat stability. Plant Physiol. 1995 Sep;109(1):245–251. doi: 10.1104/pp.109.1.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dickinson D. B., Preiss J. Presence of ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize Endosperm. Plant Physiol. 1969 Jul;44(7):1058–1062. doi: 10.1104/pp.44.7.1058. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Govons S., Vinopal R., Ingraham J., Preiss J. Isolation of mutants of Escherichia coli B altered in their ability to synthesize glycogen. J Bacteriol. 1969 Feb;97(2):970–972. doi: 10.1128/jb.97.2.970-972.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hannah L. C., Nelson O. E., Jr Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize. Biochem Genet. 1976 Aug;14(7-8):547–560. doi: 10.1007/BF00485834. [DOI] [PubMed] [Google Scholar]
- Iglesias A. A., Barry G. F., Meyer C., Bloksberg L., Nakata P. A., Greene T., Laughlin M. J., Okita T. W., Kishore G. M., Preiss J. Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli. J Biol Chem. 1993 Jan 15;268(2):1081–1086. [PubMed] [Google Scholar]
- Lin T. P., Caspar T., Somerville C. R., Preiss J. A Starch Deficient Mutant of Arabidopsis thaliana with Low ADPglucose Pyrophosphorylase Activity Lacks One of the Two Subunits of the Enzyme. Plant Physiol. 1988 Dec;88(4):1175–1181. doi: 10.1104/pp.88.4.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin T. P., Caspar T., Somerville C., Preiss J. Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity. Plant Physiol. 1988 Apr;86(4):1131–1135. doi: 10.1104/pp.86.4.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Morell M., Bloom M., Preiss J. Affinity labeling of the allosteric activator site(s) of spinach leaf ADP-glucose pyrophosphorylase. J Biol Chem. 1988 Jan 15;263(2):633–637. [PubMed] [Google Scholar]
- Müller-Röber B., Sonnewald U., Willmitzer L. Inhibition of the ADP-glucose pyrophosphorylase in transgenic potatoes leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO J. 1992 Apr;11(4):1229–1238. doi: 10.1002/j.1460-2075.1992.tb05167.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakata P. A., Greene T. W., Anderson J. M., Smith-White B. J., Okita T. W., Preiss J. Comparison of the primary sequences of two potato tuber ADP-glucose pyrophosphorylase subunits. Plant Mol Biol. 1991 Nov;17(5):1089–1093. doi: 10.1007/BF00037149. [DOI] [PubMed] [Google Scholar]
- Okita T. W. Is there an alternative pathway for starch synthesis? Plant Physiol. 1992 Oct;100(2):560–564. doi: 10.1104/pp.100.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okita T. W., Nakata P. A., Anderson J. M., Sowokinos J., Morell M., Preiss J. The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase. Plant Physiol. 1990 Jun;93(2):785–790. doi: 10.1104/pp.93.2.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Smith-White B. J., Preiss J. Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources. J Mol Evol. 1992 May;34(5):449–464. doi: 10.1007/BF00162999. [DOI] [PubMed] [Google Scholar]
- Tsai C. Y., Nelson O. E. Starch-deficient maize mutant lacking adenosine dephosphate glucose pyrophosphorylase activity. Science. 1966 Jan 21;151(3708):341–343. doi: 10.1126/science.151.3708.341. [DOI] [PubMed] [Google Scholar]