Salt-induced folding of a rabbit muscle pyruvate kinase intermediate at alkaline pH

The effect of alkaline denaturation on the structural and functional charac teristics of rabbit muscle pyruvate kinase (PK) was investigated using enzy matic activity measurements and a combination of optical methods such as ci rcular 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 str ong binding to the hydrophobic dye ANS. This intermediate retains the enzym atic activity and corresponds to a dimeric state of the molecule. Above pH 10.5, a sudden fall in the spectral properties and enzymatic activity occur s suggesting the dissociation of the molecule followed by unfolding at very high pH. Addition of salts such as NaCl, KCl, and Na2SO4 to the alkali-ind uced state induces both secondary and tertiary structure to a level equival ent to that of native tetramer (salt-induced state). Chemical- and temperat ure-induced un folding of the alkali-induced state as well as the salt-indu ced 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 o f denaturants during unfolding. The observations presented in this paper su ggest that the native pyruvate kinase tetramer dissociates to an active dim er around pH 10.5 and further to inactive monomer before attaining a comple tely unfolded monomeric conformation.