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ADP-Glucose Pyrophosphorylase from Potato Tubers.
Site-Directed Mutagenesis Studies of the Regulatory Sites1
Miguel A. Ballicora,
Yingbin Fu,
Natasha M. Nesbitt, and
Jack Preiss*
Department of Biochemistry, Michigan State University, East
Lansing, Michigan 48824
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.
1
This work was supported in part by Department of
Energy grant no. DE-FG02-93ER20121.
*
Corresponding author; e-mail preiss{at}pilot.msu.edu; fax
1-517-353-9334.
Plant Physiol. (1998) 118: 265-274
Copyright Clearance Center: 0032-0889/98/118//10
© 1998 American Society of Plant Physiologists
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