Site-directed mutagenesis of Lysine{sup 382}, the activator-binding site, of ADP-Glucose pyrophosphorylase from Anabaena PCC 6120
- Michigan State Univ., East Lansing, MI (United States)
Previous studies have shown that a highly conserved lysyl residue (Lys{sup 419}) near the C-terminus of Anabaena ADP-glucose pyrophosphorylase is involved in the binding of 3-P-glycerate, the allosteric activator. Phosphopyridoxylation of the K419R mutant enzyme modified another conserved lysyl residue (Lys{sup 382}), suggesting that this residue might be also located within the activator-binding site. Site-directed mutagenesis of Lys{sup 382} of the Anabaena enzyme was performed to determine the role of this residue. Replacing Lys{sup 382} with either arginine, alanine, or glutamine produced mutant enzymes with apparent affinities for 3-P-glycerate 10-160-fold lower than that of the wild-type enzyme. The glutamic acid mutant enzyme was inhibited by 3-P-glycerate. These mutations had lesser impact on the kinetic constants for the substrates and inhibitor, P{sub i}, and on the thermal stability. These results indicate that both the charge and size of the residue at position 382 influence the binding of 3-P-glycerate. Site-directed mutagenesis was also performed to obtain a K382R-K419R double mutant. The apparent affinity for 3-P-glycerate of this double-mutant enzyme was 104-fold lower than that of the wild-type enzyme, and the specificity for activator of this mutant enzyme was altered. The K382R-K419R enzyme could not be phosphopyridoxylated, suggesting that other lysine residues are not involved in the binding of 3-P-glycerate. 32 refs., 2 figs., 3 tabs.
- OSTI ID:
- 468992
- Journal Information:
- Biochemistry (Eaton), Vol. 35, Issue 9; Other Information: PBD: 5 Mar 1996
- Country of Publication:
- United States
- Language:
- English
Similar Records
Site-directed mutagenesis of lysine-329 of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum
Mutagenesis of the potato ADPglucose pyrophosphorylase and characterization of an allosteric mutant defective in 3-phosphoglycerate activation