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

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. 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 I{sub 2} 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 wildtype 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 studiesmore » to be important in the binding of 3-PGA. The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP. 28 refs., 3 figs., 1 tab.« less

Authors:
; ;  [1]
  1. Washington State Univ., Pullman, WA (United States)
Publication Date:
OSTI Identifier:
494256
DOE Contract Number:  
FG06-94ER20160
Resource Type:
Journal Article
Journal Name:
Plant Physiology (Bethesda)
Additional Journal Information:
Journal Volume: 112; Journal Issue: 3; Other Information: PBD: Nov 1996
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GLYCOGEN; BIOSYNTHESIS; PHOSPHOTRANSFERASES; ENZYME ACTIVITY; ASPARTIC ACID; ATP; DNA; GLUCOSE; MUTANTS; LYSINE; AMINO ACID SEQUENCE

Citation Formats

Greene, T.W., Woodbury, R.L., and Okita, T.W. Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase. United States: N. p., 1996. Web. doi:10.1104/pp.112.3.1315.
Greene, T.W., Woodbury, R.L., & Okita, T.W. Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase. United States. doi:10.1104/pp.112.3.1315.
Greene, T.W., Woodbury, R.L., and Okita, T.W. Fri . "Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase". United States. doi:10.1104/pp.112.3.1315.
@article{osti_494256,
title = {Aspartic acid 413 is important for the normal allosteric functioning of ADP-glucose pyrophosphorylase},
author = {Greene, T.W. and Woodbury, R.L. and Okita, T.W.},
abstractNote = {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. 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 I{sub 2} 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 wildtype 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. The kinetic properties of M27 corroborate the importance of this region in the allosteric regulation of a higher-plant AGP. 28 refs., 3 figs., 1 tab.},
doi = {10.1104/pp.112.3.1315},
journal = {Plant Physiology (Bethesda)},
number = 3,
volume = 112,
place = {United States},
year = {1996},
month = {11}
}