R-State AMP Complex Reveals Initial Steps of the Quaternary Transition of Fructose-1,6-bisphosphatase

• Published in: The Journal of biological chemistry 2005-05, Vol.280 (20), p.19737-19745
• Main Authors: Iancu, Cristina V., Mukund, Susmith, Fromm, Herbert J., Honzatko, Richard B.
• Format: Article
• Language: English
• Subjects: Adenosine Monophosphate - chemistry; Adenosine Monophosphate - metabolism; Amino Acid Substitution; Base Sequence; Binding Sites - genetics; Crystallography, X-Ray; DNA, Bacterial - genetics; Enzyme Stability; Escherichia coli - enzymology; Escherichia coli - genetics; Fructose-Bisphosphatase - antagonists & inhibitors; Fructose-Bisphosphatase - chemistry; Fructose-Bisphosphatase - genetics; Fructose-Bisphosphatase - metabolism; Hydrogen Bonding; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Protein Conformation; Protein Structure, Quaternary; Protein Structure, Tertiary; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Static Electricity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M501011200

AMP transforms fructose-1,6-bisphosphatase from its active R-state to its inactive T-state; however, the mechanism of that transformation is poorly understood. The mutation of Ala54 to leucine destabilizes the T-state of fructose-1,6-bisphosphatase. The mutant enzyme retains wild-type levels of activity, but the concentration of AMP that causes 50% inhibition increases 50-fold. In the absence of AMP, the Leu54 enzyme adopts an R-state conformation nearly identical to that of the wild-type enzyme. The mutant enzyme, however, grows in two crystal forms in the presence of saturating AMP. In one form, the AMP-bound tetramer is in a T-like conformation, whereas in the other form, the AMP-bound tetramer is in a R-like conformation. The latter reveals conformational changes in two helices due to the binding of AMP. Helix H1 moves toward the center of the tetramer and displaces Ile10 from a hydrophobic pocket. The displacement of Ile10 exposes a hydrophobic surface critical to interactions that stabilize the T-state. Helix H2 moves away from the center of the tetramer, breaking hydrogen bonds with a buried loop (residues 187–195) in an adjacent subunit. The same hydrogen bonds reform but only after the quaternary transition to the T-state. Proposed here is a model that accounts for the quaternary transition and cooperativity in the inhibition of catalysis by AMP.