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


CONCLUSIONS
Mimics of the substrate inhibition of fructose-1,6-bisphosphatase may provide a future option for combatting both postprandial and fasting hyperglycemia.
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Joel L
JL
Asenjo
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Heide C
HC
Ludwig
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Cristian A
CA
Droppelmann
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Juan G
JG
Cárcamo
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Ilona I
II
Concha
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile.
Alejandro J
AJ
Yáñez
Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, CNRS, Aix-Marseille Université, Marseilles, France.
María L
ML
Cárdenas
Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, CNRS, Aix-Marseille Université, Marseilles, France.
Athel
A
Cornish-Bowden
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Valdivia, Chile. Electronic address: jslebe@uach.cl.
Juan C
JC
Slebe
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D055162:Biocatalysis; D006597:Fructose-Bisphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D005635:Fructosediphosphates / Q000737:chemistry; D007668:Kidney / Q000201:enzymology*; D008969:Molecular Sequence Data; D021122:Protein Subunits; D013379:Substrate Specificity; D013552:Swine
D005635:Fructosediphosphates; D021122:Protein Subunits; D006597:Fructose-Bisphosphatase
Affinity site; FBPase; Fructose-1,6-bisphosphate; Gluconeogenesis; Substrate inhibition; Subunit interaction