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Probing the Roles of Arginines 26 and 38 in the Allosteric Regulation of ADPGlucose Pyrophosphorylase from Thermus thermophilus
Author(s) -
Botero Fabian,
Truc Teddy,
Boukhari Mokhtar,
Meyer Christopher R
Publication year - 2011
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.25.1_supplement.765.6
Subject(s) - allosteric regulation , thermus thermophilus , biochemistry , enzyme , chemistry , allosteric enzyme , mutant , activator (genetics) , gene , escherichia coli
ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate‐limiting step in glucan synthesis. Given the role this allosteric enzyme plays in the production of biodegradable and renewable carbon, it is an attractive target for protein engineering. Somewhat unique to the bacterial enzymes, the Thermus thermophilus ADPG PPase was found to be activated by G6P as well as by F6P and FBP. Based on alignment and molecular modeling studies, R26 and R38 were proposed to play a role in allosteric regulation of this thermophillic ADPG PPase. As a first step, the R26A and R38A enzymes were generated and purified for characterization. The R26A and R38A enzymes both displayed a significant decrease in Vmax compared to the wild‐type enzyme in the absence and presence of activators. In the absence of activators, both enzymes also displayed a modest decrease in the apparent affinity for substrates compared to the wild‐type enzyme. In contrast to the wild‐type enzyme, the presence of the activators resulted in an increase in apparent affinity for the substrate ATP for both altered proteins. The most dramatic and specific effect of the mutations was the ~8‐fold decrease in apparent affinity for the activator G6P displayed by the R26A enzyme. The diminution of Vmax is consistent with a role for these arginines in stabilizing an active conformation of the enzyme. In addition, R26 appears to play a role in the binding of G6P, likely via an electrostatic interaction with the phosphate moiety. Further kinetic studies and generation and characterization of the double mutant are underway.