31P nuclear magnetic resonance spectroscopy studies of substrate and product binding to fructose-1,6-bisphosphatase.
The enzymatic hydrolysis of fructose 1,6-bisphosphate (Fru-1,6-P2) to fructose 6-phosphate (Fru-6-P) and inorganic phosphate (Pi), which is catalyzed by fructose-1,6-bisphosphatase, has been studied by 31P nuclear magnetic resonance spectroscopy (NMR). At pH 7.5 and 15 degrees C, the equilibrium constant for the central complex K'eq = [E.Fru-6-P.Pi]/[E.Fru-1,6-P2.H2O] is about 2. This observation is in harmony with results obtained with a number of Bi Bi enzyme systems for the determination of K'eq in which a variety of experimental techniques were used (Knowles, J.R. (1980) Annu. Rev. Biochem. 49, 877-919). Significant changes in 31P NMR chemical shifts were observed for both the substrate, Fru-1,6-P2, and the product, Fru-6-P, when bound to the enzyme relative to ligand free in solution. The chemical shifts of the substrate and product were altered further in the presence of Mg2+, the catalytic divalent metal ion. The chemical shifts caused by the addition of metal ion can be reversed in the presence of trans-1,2-diaminocyclohexane- N,N,N',N'-tetraacetic acid (CDTA) or AMP. In the presence of the metal ion chelator or the nucleotide, the substrate had a chemical shift that was about the same as that observed in the absence of metal ion. On the basis of these observations we suggest that AMP and CDTA exhibit similar effects, i.e. they both remove the catalytic metal ion from the enzyme. This finding is supportive of the suggestion (Scheffler, J. E., and Fromm, H.J. (1986) Biochemistry 25, 6659-6665; Liu, F., and Fromm, H.J. (1990) J. Biol. Chem. 265, 7401-7406) that the role of AMP in the regulation of fructose-1,6-bisphosphatase is to prevent binding of the divalent metal activator to the enzyme.
Liu
F
F
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Fromm
H J
HJ
D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D004492:Edetic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D010759:Phosphorus Isotopes; D011817:Rabbits; D013379:Substrate Specificity
D002413:Cations, Divalent; D010759:Phosphorus Isotopes; D000249:Adenosine Monophosphate; C005390:CDTA; D004492:Edetic Acid; D006597:Fructose-Bisphosphatase