Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: binding of substrate induces a second class of site with lowered affinity and catalytic activity.

Joel L Asenjo, Heide C Ludwig, Cristian A Droppelmann, Juan G Cárcamo, Ilona I Concha, Alejandro J Yáñez, María L Cárdenas, Athel Cornish-Bowden, Juan C Slebe

Index: Biochim. Biophys. Acta 1840(6) , 1798-807, (2014)

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Abstract

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.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.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.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.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.