Regulation of rabbit liver fructose-1,6-bisphosphatase by metals, nucleotides, and fructose 2,6-bisphosphate as determined from fluorescence studies.
The fluorescent nucleotide analogue formycin 5'-monophosphate (FMP) inhibits rabbit liver fructose-1,6-bisphosphatase (I50 = 17 microM, Hill coefficient = 1.2), as does the natural regulator AMP (I50 = 13 microM, Hill coefficient = 2.3), but exhibits little or no cooperativity of inhibition. Binding of FMP to fructose-1,6-bisphosphatase can be monitored by the increased fluorescence emission intensity (a 2.7-fold enhancement) or the increased fluorescence polarization of the probe. A single dissociation constant for FMP binding of 6.6 microM (4 sites per tetramer) was determined by monitoring fluorescence intensity. AMP displaces FMP from the enzyme as evidenced by a decrease in FMP fluorescence and polarization. The substrates, fructose 6-phosphate and fructose 1,6-bisphosphate, and inhibitors, methyl alpha-D-fructofuranoside 1,6-bisphosphate and fructose 2,6-bisphosphate, all increase the maximal fluorescence of enzyme-bound FMP but have little or no effect on FMP binding. Weak metal binding sites on rabbit liver fructose-1,6-bisphosphatase have been detected by the effect of Zn2+, Mn2+, and Mg2+ in displacing FMP from the enzyme. This is observed as a decrease in FMP fluorescence intensity and polarization in the presence of enzyme as a function of divalent cation concentration. The order of binding by divalent cations is Zn2+ = Mn2+ greater than Mg2+, and the Kd for Mn2+ displacement of FMP is 91 microM. Methyl alpha-D-fructofuranoside 1,6-bisphosphate, as well as fructose 6-phosphate and inorganic phosphate, enhances metal-mediated FMP displacement from rabbit liver fructose-1,6-bisphosphatase.(ABSTRACT TRUNCATED AT 250 WORDS)
D000249:Adenosine Monophosphate / Q000494:pharmacology*; D000818:Animals; D002413:Cations, Divalent; D005573:Formycins / Q000494:pharmacology; D006597:Fructose-Bisphosphatase / Q000378:metabolism*; D005635:Fructosediphosphates / Q000494:pharmacology*; D006598:Hexosediphosphates / Q000494:pharmacology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D011817:Rabbits; D012265:Ribonucleotides / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D015032:Zinc / Q000494:pharmacology
D002413:Cations, Divalent; D005573:Formycins; D005635:Fructosediphosphates; D006598:Hexosediphosphates; D012265:Ribonucleotides; C021683:formycin 5'-phosphate; D000249:Adenosine Monophosphate; D008345:Manganese; D006597:Fructose-Bisphosphatase; D008274:Magnesium; D015032:Zinc; C029063:fructose-1,6-diphosphate