The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase.
The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.
D000249:Adenosine Monophosphate / Q000378:metabolism*; D000818:Animals; D005634:Fructose-Bisphosphate Aldolase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000378:metabolism*; D006598:Hexosediphosphates / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D011487:Protein Conformation; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism
D005635:Fructosediphosphates; D006598:Hexosediphosphates; D000249:Adenosine Monophosphate; D008345:Manganese; C027652:fructose 2,6-diphosphate; D005634:Fructose-Bisphosphate Aldolase; D008274:Magnesium; D015032:Zinc