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The role of Aspartate 141 in the regulation of the Agrobacterium tumefaciens ADP‐Glc Pyrophosphorylase
Author(s) -
Patel Hiralben,
GonzalezMartin Laura,
Renta Vincent,
Ballicora Miguel
Publication year - 2016
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.30.1_supplement.834.22
Subject(s) - allosteric regulation , protein subunit , enzyme , biochemistry , agrobacterium tumefaciens , mutant , pyrophosphate , chemistry , stereochemistry , biology , gene , transgene
The main regulatory step of the glycogen and starch metabolic pathways takes place at the level of the synthesis of ADP‐glucose (ADP‐Glc), which is the first committed step and it is catalyzed by ADP‐Glc Pyrophosphorylase (ADP‐Glc PPase). This is an allosterically regulated enzyme, which catalyzes the conversion of ATP and glucose‐1‐phosphate to ADP‐Glc and inorganic pyrophosphate in the presence of Mg 2 . In previous studies, it was observed that an inter‐subunit conformational change in the potato tuber ADP‐Glc PPase may play a role in the allosteric regulation of the enzyme. Removal of a disulfide bond involving Cys12 in the small subunit, either by mutation or by reduction resulted in a more active enzyme (Ballicora et al, 2000 , J. Biol. Chem . 275 :1315–1320), indicating that the inter‐subunit interaction between α subunits is an important part of the allosteric mechanism (Jin, X. et al, 2005, EMBO J. 24: 694–704). In the structure of the Agrobacterium tumefaciens ADP‐Glc PPase, we found that Asp141 is a candidate to interact with Arg11 of the opposing subunit (Cupp‐Vickery, et al. 2008, Biochemistry 47 :12795–801). The extreme N‐terminal Arg5 and Arg11 are quite clearly involved in activation by pyruvate (Pyr) as it has been demonstrated before (Gomez‐Casati et al. Biochem , 40(34) ,2001, pp. 10169–10178). To evaluate the hypothesis that of Asp 141 is involved in regulation, we mutated it to Ala. D141A was successfully expressed and purified to near homogeneity. This mutant enzyme exhibited reduced V max values in the absence of activators (Pyr and Fru6P), and lower apparent affinity for ATP and Fru6P. In the presence of Fru6P, D141A enzyme exhibited only ~ 4.3% of the wild‐type activity, which indicates that it virtually insensitive to Fru6P. This mutant enzyme also displayed 2.5‐fold reduced apparent affinity to the activator Fru6P when compared to the wild‐type enzyme. Our results suggest a unique functional role of D141 in regulation, possibly by interacting with R11 in the opposing subunit. Support or Funding Information National Science Foundation (NSF)