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Probing the Molecular Basis for Heat Stability and Substrate and Activator Specificity for Thermus thermophilus ADPGlucose Pyrophosphorylase
Author(s) -
Doshi Dhaval,
Bhatt Shrey,
Harkison Nicul,
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.lb235
Subject(s) - allosteric regulation , enzyme , gtp' , thermus thermophilus , activator (genetics) , chemistry , biochemistry , mutant , nucleotide , wild type , enzyme assay , enzyme activator , receptor , escherichia coli , gene
ADPGlucose Pyrophosphorylase catalyzes the rate‐limiting step in glucan synthesis. The heat stable T. thermus enzyme is activated by FBP, F6P, and G6P. In order to investigate the molecular basis for heat stability, three unique prolines (P100, 122, 195) have been changed to alanine, and the single and triple mutant proteins purified and characterized. All of the single exhibited reduced Vmax values compared to wild‐type but no significant change in heat stability. The triple mutant had ~30 fold less activity than wild‐type as well as altered apparent affinity for substrates. The wild‐type enzyme was able to utilize CTP, GTP, and UTP in place of ATP to a limited extent, with the alternative nucleotides having Vmax values 15, 3, and 0.1% of wild‐type, respectively. In the presence of UTP, the enzyme displayed higher fold activation. The S0.5 values were increased for these nucleotides as well ranging from a ~20 fold increase for GTP to a 1.5 fold increase for CTP. Molecular modeling indicates involvement of K376 in the allosteric site. In contrast to wild‐type, the K376A enzyme displayed inhibition by the substrate ATP as well as a lower Vmax in the absence of activator. Preliminary data collected at subsaturating ATP concentrations indicates that the altered enzyme has decreased apparent affinity for activators as well as lower fold activation. Supported in part by NSF Grant 0448676.