Unfolding pathway in red kidney bean acid phosphatase is dependent on ligand binding.
Structural basis for ligand-induced protein stabilization was investigated in the case of an acid phosphatase (red kidney bean purple acid phosphatase (KBPAP)) from red kidney bean. Phosphate, a physiological ligand, increases the stability against solvent denaturation by 3.5 kcal/mol. Generality of phosphate stabilization was shown by similar effects 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 the dissociation constant estimated by conventional methods. The guanidinium chloride-mediated denaturation of KBPAP was monitored by several structural and functional parameters viz. activity, tryptophan fluorescence, 8-anilinonaphthalene 1-sulfonic acid binding, circular dichroism, and size exclusion chromatography, in the presence and absence of 10 mm phosphate. In the presence of phosphate, profiles of all the parameters shift to a higher guanidinium chloride concentration. Noncoincidence of these profiles in the absence of phosphate indicates multistate unfolding pathway for KBPAP; however, in the presence of phosphate, KBPAP unfolds with a single intermediate. Based on the crystal structure, we propose that the Arg258 may have an important role to play in stabilization mediated by phosphate.
Cashikar
A G
AG
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Rao
N M
NM
D000135:Acid Phosphatase / Q000737:chemistry* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D002942:Circular Dichroism; D007887:Fabaceae / Q000201:enzymology*; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008623:Mercaptoethanol / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D010946:Plants, Medicinal; D011193:Potassium Iodide; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D005456:Fluorescent Dyes; D006146:Guanidines; D008024:Ligands; D010710:Phosphates; D011193:Potassium Iodide; C022571:adenosine 5'-O-(3-thiotriphosphate); D008623:Mercaptoethanol; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000135:Acid Phosphatase; D019791:Guanidine