Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH.
The effect of alkaline denaturation on the structural and functional characteristics of rabbit muscle pyruvate kinase (PK) was investigated using enzymatic activity measurements and a combination of optical methods such as circular dichroism, fluorescence, and ANS binding. At a critical pH, 10.5, PK exists in an intermediate state (alkaline unfolded state) with predominant secondary structure along with some of the tertiary interactions and a strong binding to the hydrophobic dye ANS. This intermediate retains the enzymatic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occurs suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCI, and Na2SO4 to the alkali-induced state induces both secondary and tertiary structure to a level equivalent to that of native tetramer (salt-induced state). Chemical- and temperature-induced unfolding of the alkali-induced state as well as the salt-induced refolded state of PK reveal the presence of intermediate conformations in the unfolding pathway. The unfolding transition curves are noncoinciding and noncooperative along with ANS binding at intermediate concentrations of denaturants during unfolding. The observations presented in this paper suggest that the native pyruvate kinase tetramer dissociates to an active dimer around pH 10.5 and further to inactive monomer before attaining a completely unfolded monomeric conformation.
Edwin
F
F
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India. fedwin@biochem.umass.edu
Jagannadham
M V
MV
D000818:Animals; D006863:Hydrogen-Ion Concentration; D009132:Muscles / Q000201:enzymology*; D017510:Protein Folding; D011770:Pyruvate Kinase / Q000737:chemistry*; D011817:Rabbits; D012492:Salts / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D012492:Salts; D011770:Pyruvate Kinase