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(The FASEB Journal. 2007;21:806.6)
© 2007 FASEB
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806.6

Probing the Activator Specificity of Rhodospirillum rubrum ADPGlucose Pyrophosphorylase

Jared Wiig1, Dhaval Doshi1, Adeel Amer1, Chintan Amin1, Piranid Charoensak1, Andrew Orry2, Ruben Abagyan2 and Christopher R. Meyer1

1 Chemistry and Biochemistry, California State University, Fullerton, 800 N. State College Blvd., Fullerton, CA, 92834,
2 Molsoft LCC, 3366 North Torrey Pines Court, Suite 300, La Jolla, CA, 92037

ABSTRACT

The allosteric enzyme ADPGlucose Pyrophosphorylase (ADPG PPase) catalyzes the rate-limiting step in glucan biosynthesis. Typical bacterial enzymes are activated by sugar phosphates and inhibited by AMP and/or phosphate. The Rhodospirillum rubrum (Rs.r.) enzyme is activated only by pyruvate and has no inhibitors. Previous work demonstrated that a chimeric enzyme comprised of the Rs.r. N-terminus and the E. coli C-terminus (pSM3) could be activated by FBP if a small region of the N-terminus was replaced with E. coli residues. Focusing on this region, molecular modeling of the Rs.r. structure has revealed the apparent disruption of a salt bridge present in other ADPG PPases which links the N- and C-terminal domains. In silico docking experiments indicate that R50 and R84 are involved in pyruvate binding. Positions in the N-terminus were probed by mutagenesis. The pSM3 Y49F enzyme showed strong activation by pyruvate and inhibition by FBP and F6P. The Rs.r. R50A and R84A enzymes displayed low specific activity, reduced apparent affinity for pyruvate, and inhibition by FBP and F6P. The purified R84K enzyme was substantially activated by pyruvate. Multiple mutations appear to be necessary to allow activation by sugar phosphates, although inhibition can be attained by a single point mutation. Complete characterization of the R50A-R84A, R50K, and R84K altered enzymes is underway.

Supported in part by NSF grant 0448676.





This Article
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