1. Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: binding of substrate induces a second class of site with lowered affinity and catalytic activity.
- Author
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Asenjo JL, Ludwig HC, Droppelmann CA, Cárcamo JG, Concha II, Yáñez AJ, Cárdenas ML, Cornish-Bowden A, and Slebe JC
- Subjects
- Animals, Base Sequence, Binding Sites, Fructose-Bisphosphatase antagonists & inhibitors, Fructose-Bisphosphatase metabolism, Fructosediphosphates chemistry, Molecular Sequence Data, Protein Subunits, Substrate Specificity, Swine, Biocatalysis, Fructose-Bisphosphatase chemistry, Kidney enzymology
- Abstract
Background: Fructose-1,6-bisphosphatase, a major enzyme of gluconeogenesis, is inhibited by AMP, Fru-2,6-P2 and by high concentrations of its substrate Fru-1,6-P2. The mechanism that produces substrate inhibition continues to be obscure., Methods: Four types of experiments were used to shed light on this: (1) kinetic measurements over a very wide range of substrate concentrations, subjected to detailed statistical analysis; (2) fluorescence studies of mutants in which phenylalanine residues were replaced by tryptophan; (3) effect of Fru-2,6-P2 and Fru-1,6-P2 on the exchange of subunits between wild-type and Glu-tagged oligomers; and (4) kinetic studies of hybrid forms of the enzyme containing subunits mutated at the active site residue tyrosine-244., Results: The kinetic experiments with the wild-type enzyme indicate that the binding of Fru-1,6-P2 induces the appearance of catalytic sites with lower affinity for substrate and lower catalytic activity. Binding of substrate to the high-affinity sites, but not to the low-affinity sites, enhances the fluorescence emission of the Phe219Trp mutant; the inhibitor, Fru-2,6-P2, competes with the substrate for the high-affinity sites. Binding of substrate to the low-affinity sites acts as a "stapler" that prevents dissociation of the tetramer and hence exchange of subunits, and results in substrate inhibition., Conclusions: Binding of the first substrate molecule, in one dimer of the enzyme, produces a conformational change at the other dimer, reducing the substrate affinity and catalytic activity of its subunits., General Significance: Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
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