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Probing the Role of Aspartate‐378 in the Regulation of ADPGlucose Pyrophosphorylase from Rhodobacter sphaeroides
Author(s) -
Fernandez Paola,
Matsui Mikiko,
Orry Andrew,
Meyer Christopher R
Publication year - 2009
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.23.1_supplement.503.6
Subject(s) - allosteric regulation , enzyme , chemistry , biochemistry , ligand (biochemistry) , phosphofructokinase 2 , stereochemistry , receptor
ADP‐glucose pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step in glucan synthesis. The R. sphaeroides ADPG PPase is activated by F6P, pyruvate, and FBP and inhibited by PEP and phosphate. The structure of the similar A. tumefaciens ADPG PPase (2008 Biochemistry 47 , 4439‐4451) indicates that a salt‐bridge connecting R33 and D378 is involved in formation of an allosteric site. Mutations of R33 resulted in desensitization to effectors. The D378A,N, and E enzymes were generated and characterized to probe the role of size, shape and charge. Without effectors, all of the enzymes had a lower apparent affinity for ATP while the D378A and D378E enzymes exhibited a ten fold lower and two‐fold higher V max compared to wild‐type, respectively. In contrast to wild‐type and D378N, E enzymes, the D378A did not exhibit K‐type activation. All of the altered enzymes exhibited both lower fold activation by F6P, FBP, and pyruvate and a higher apparent affinity for activators, consistent with a change in the allosteric site resulting in an altered enzyme form which allows for tighter ligand binding to the exposed R33. I n silico ligand docking studies are in progress. Supported in part by NSF Grant 0448676.