A fluorescence study of ligand-induced conformational changes in cytosolic fructose-1,6-bisphosphatase from germinating castor oil seeds

摘要

The intrinsic fluorescence of homogeneous castor oil seed cytosolic fructose-1,6-bisphosphatase (FBPase_c) was used as an indicator of conformational changes due to ligand binding. Binding of the substrate and the inhibitor fructose-2,6-bisphosphate (F-2,6-P_2) was quantitatively compared to their respective kinetic effects on enzymatic activity. There are two distinct types of substrate interaction with FBPase_c, corresponding to catalytic and inhibitory binding, respectively. Inhibitory substrate binding shares several characteristics with F-2,6-P_2 binding which indicates that both ligands bind at the same site. However, F-2,6-P_2 does not prevent fluorescence transitions attributed to catalytic substrate binding. The marked synergistic inhibition of FBPase_c by AMP and F-2, 6-P_2 appears to arise via AMP's promotion of F-2, 6-P_2 binding. Based on the X-ray crystal structure of porcine kidney FBPase our modelling studies suggest the existence of a distinct F-1,6-P_2/F-2,6-P_2 inhibitory binding site which partially overlaps with the enzyme's catalytic site. We propose that a pronounced allosteric transition mediated by AMP binding increases access of F-1, 6-P_2 and F-2, 6-P_2 to this common inhibitory binding site.