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Characterization of Thermus thermophilus ADPGlucose Pyrophosphorylase
Author(s) -
Gonzalez Jacob,
Watanabe Go,
Orry Andrew,
Abagyan Ruben,
Meyer Christopher R.
Publication year - 2007
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.21.5.a642-d
Subject(s) - thermus thermophilus , enzyme , allosteric regulation , gtp' , chemistry , biochemistry , specific activity , recombinant dna , escherichia coli , gene
ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes a key step in glucan synthesis and is regulated by allosteric metabolites. Little is known about thermophillic forms of this enzyme. We have successfully expressed and purified the recombinant enzyme from T. thermophilus HB27 and collected kinetic data at 75°C. This has now been extended to include 37°C and alternate nucleotide specificity. We have also identified three unique prolines in the sequence compared to other ADPG PPases which may provide structural rigidity and account for heat stability. The V max at 37°C was ~4‐fold lower then measured at 75°C. The S 0.5 for ATP and Mg with and without activators (G6P, FBP, F6P) were in fair agreement with the results obtained at 75°C. The S 0.5 for GIP in the presence of the activators were decreased ~3‐fold and the A 0.5 for the activators were ~2‐fold increased. The V‐type activation (~2–3‐fold) was similar for the activators at both temperatures. The specific activity for CTP, GTP, UTP, and TTP were determined to be 0.6%, 0.3%, 0.15%, and 0.001%, respectively, of the activity with ATP as a substrate. Interestingly, the fold‐activation increased dramatically for CTP and UTP, to ~9 and ~30‐fold by FBP, respectively. The P100A, P122A, and P195A mutations were successfully generated by site‐directed mutagenesis and characterization is in progress. Supported in part by NSF grant 0448676 and NIH MARC 2 T34‐GM008612‐09.