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Characterization of the Thermotoga maritima ADP‐glucose pyrophosphorylase: Both glgC and glgD Subunits are Required for Optimal Activity and Allosteric regulation
Author(s) -
Matsui Mikiko,
Guandique Erick,
Tran Margaret,
Nisperos Sean,
Orry Andrew,
Meyer Christopher
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.835.10
Subject(s) - allosteric regulation , chemistry , biochemistry , enzyme
The T. maritima ADP‐glucose pyrophosphorylase (ADPG PPase) is comprised of both glgC and glgD subunits. Analytical ultracentrifugation, size‐exclusion chromatography, and native‐PAGE analyses all indicate that glgC and glgD exist as monomers that oligomerize to form a glgD/C heterotetramer. Functional assays revealed negligible glgD activity alone; however, glgC showed ~20‐fold stimulation when combined with glgD. The kinetic parameters for glgC alone and the glgD/C complex were determined. glgC was insensitive to a screen of 15 known ADPG PPase effector molecules, but glgD/C exhibited 2.2‐fold activation by FBP. The glgD/C S 0.5 values for ATP, G1P, and Mg 2+ showed increased apparent affinities than glgC alone and the specific activity was 3.2‐fold higher than glgC at 37°C. The glgD/C S 0.5 values for ATP, G1P, and Mg 2+ also showed increased apparent affinities and a 2.7‐fold higher specific activity than glgC alone at 75°C. Both glgC K26A and D244N studies resulted in significantly decreased glgC activity, indicating that these two residues are important for catalysis. Supported in part by NSF Grant 0448676.