Modulation of the interaction between aldolase and glycerol-phosphate dehydrogenase by fructose phosphates.
Kinetics of fructose-1,6-disphosphate aldolase (EC 4.1.2.13) catalyzed conversion of fructose phosphates was analyzed by coupling the aldolase reactions to the metabolically sequential enzyme, glycerol-3-phosphate dehydrogenase (EC 1.1.1.8), which interacts with aldolase. At low enzyme concentration poly(ethylene glycol) was added to promote complex formation of aldolase and glycerol-phosphate dehydrogenase resulting in a 3-fold increase in KM of fructose-1,6-bisphosphate and no change in Vmax. Kinetic parameters for fructose-1-phosphate conversion changed inversely upon complex formation: Vmax increased while KM remained unchanged. Gel penetration and ion-exchange chromatographic experiments showed positive modulation of the interaction of aldolase and dehydrogenase by fructose-1,6-bisphosphate. The dissociation constant of the heterologous enzyme complex decreased 10-fold in the presence of this substrate. Fructose-1-phosphate or dihydroxyacetone phosphate had no effect on the dissociation constant of the aldolase-dehydrogenase complex. In addition, titration of fluorescein-labelled glycerol-phosphate dehydrogenase with aldolase indicated that both fructose-1,6-bisphosphate and fructose-2,6-biphosphate enhanced the affinity of aldolase to glycerol-phosphate dehydrogenase. The results of the kinetic and binding experiments suggest that binding of the C-6 phosphate group of fructose-1,6-bisphosphate to aldolase complexed with dehydrogenase is sterically impeded while saturation of the C-6 phosphate group site increases the affinity of aldolase for dehydrogenase. The possible molecular mechanism of the fructose-1,6-bisphosphate modulated interaction is discussed.
Vértessy
B G
BG
Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Orosz
F
F
Ovádi
J
J
D000818:Animals; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005636:Fructosephosphates / Q000378:metabolism*; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D011817:Rabbits
D005636:Fructosephosphates; D005993:Glycerolphosphate Dehydrogenase; D005634:Fructose-Bisphosphate Aldolase