Characterization of nucleotide-binding site of rabbit liver fructose-1,6-bisphosphate aldolase.

Equilibfium dialyses, fluorescence quenching, fluorescence polarization, and kinetic measurements were used to characterize the interaction of nucleotides with rabbit liver aldolase B. Using an equilibrium dialysis method, 3.9 & 0.4 sites for AMP per tetrameric enzyme molecule were found with K d of 0.029 +: 0.006 mM.Kinetic measurements revealed pure competitive iahibition of aldolase B by AMP With Ki of 0.030 -t, 0.006 A blue shift and large increase in fluorescence intensity occurred upon binding a€ 8-aniline-1-naphthalenesulfonate to aldolase.Addition of nucleotides, phosphates, and substrates caused lowering of 8-aniline-lnaphthalenesfionate fluorescence.Several lines of evidence suggest that the ligands and 8-aniline-lnaphthalenesulfonate compete €or the same binding site.The changes in the fluorescence of the bound dye were used for calculation of dissociation constants for 16 nucleotides and their analogs with the enzyme.The affinity of 6'-mononucleotides decreased in the order: AMP =r IMP 2. etheno-AMP > CMP > GMP > ATP > ADP.It was demonstrated that the specificity of AMP binding is due to a multipoint attachment of the nucleotide to the enzyme.Etheno-AMP bound to aldolase exhibited fluorescence polarization, p b of 0.19 when excited at 320 n m and a corresponding angle of free rotation, w, of 32.1'.These values are close to the parameters calculated from the Perrin equation (pa = 0.21, w = 29.5")for the fluorophore attached rigidly to the spherical protein of 160,000 molecular weight.This suggests that etheno-AMP is rigidly bound to the enzyme.Iodide ions and methionine were employed as quenching agents of the fluorescence of free and enzyme-bound etheno-AMP.The quenching rate constants for free and bound nucleotide were 4.7 * 10' and 2.4 * lo* for I-, and 7.3 * lo8 and <la' M" s-I, for methionine, respectively.Thus, the adenine-binding site of liver aldolase is partially protected from the solvent.The similarity of AMP-binding to aldolase to that of mononucleotide binding to NAD domains in dehydrogenases and consequently the possible evolutionary relationship of aldolase to other glycolytic enzymes are discussed. mM.In recent years, overall folding patterns of proteins have