(The FASEB Journal. 2008;22:612.5)
© 2008 FASEB
(The FASEB Journal. 2008;22:612.5.)
© 2008 FASEB
Molecular Characterization of Thermus Thermophilus ADPGlucose Pyrophosphorylase
Jacob Gonzalez1,
Go Watanabe1,
Andrew Orry2,
Ruben Abagyan2 and
Christopher R Meyer1
1 Chemistry and Biochemistry, California State University, Fullerton, Fullerton, CA
2 Molsoft LCC, La Jolla, CA
ABSTRACT
ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes a key reaction in glucan synthesis and is regulated by allosteric metabolites. Little is known about thermophilic forms of this enzyme. We have successfully expressed and purified the recombinant Thermus thermophilus HB 27 enzyme and collected kinetic data at both 37°C and 75°C. In addition, three unique prolines in the sequence were identified compared to a related ADPG PPase, which may in part account for heat stability. Unexpectedly, the Vmax at 37°C was ~70% of that measured at 75°C. The S0.5 for ATP and Mg2+ in the absence of activators (G6P, FBP, and F6P) were in fair agreement with the results obtained at 75°C. The S0.5 for G1P in the absence of activators decreased ~5-fold, which may account for the catalytic efficiency. 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. Preliminary studies suggest P122A has some effect on the activity of Th.t ADPG PPase. Detailed heat stability tests as well as probing the effects of double and triple proline substitutions are in progress. Further, molecular modeling and in silico docking experiments have indicated a role for K43 in G6P binding and roles for P362, R79, and R306 in FBP binding. These and other sites are being probed by mutagenesis. Supported in part by NSF grant 0448676 and NIH MARC 2 T34-GM008612-09.
