UNFOLDING PATHWAY IN RED KIDNEY BEAN ACID-PHOSPHATASE IS DEPENDENT ONLIGAND-BINDING

Structural basis for ligand-induced protein stabilization was investig ated in the case of an acid phosphatase (red kidney bean purple acid p hosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological l igand, increases the stability against solvent denaturation by 3.5 kca l/mol. Generality of phosphate stabilization was shown by similar effe cts with other KBPAP ligands viz. adenosine 5'-O-(thiotriphosphate), a nonhydrolyzable ligand, and arsenate, an inhibitor. The dissociation constant of phosphate obtained from denaturation curves matches with t he dissociation constant estimated by conventional methods. The guanid inium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluore scence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroi sm, and size exclusion chromatography, in the presence and absence of 10 mM phosphate. In the presence of phosphate, profiles of all the par ameters shift to a higher guanidinium chloride concentration. Noncoinc idence of these profiles in the absence of phosphate indicates multist ate unfolding pathway for KBPAP; however, in the presence of phosphate , KBPAP unfolds with a single intermediate. Based on the crystal struc ture, we propose that the Arg(258) may have an important role to play in stabilization mediated by phosphate.