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

Abstract

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.