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

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.
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https://hal-amu.archives-ouvertes.fr/hal-01494601
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Submitted on : Thursday, March 23, 2017 - 4:44:35 PM
Last modification on : Friday, October 22, 2021 - 3:34:51 AM

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Joel L. Asenjo, Heide C. Ludwig, Cristian A. Droppelmann, Juan G. Cárcamo, Ilona I. Concha, et al.. Subunit interactions in pig-kidney fructose-1,6-bisphosphatase: Binding of substrate induces a second class of site with lowered affinity and catalytic activity. Biochimica et Biophysica Acta (BBA) - General Subjects, Elsevier, 2014, 1840 (6), pp.1798 - 1807. ⟨10.1016/j.bbagen.2013.12.027⟩. ⟨hal-01494601⟩

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