Anion-induced folding of rabbit muscle pyruvate kinase: Existence of multiple intermediate conformations at low pH

Structural and functional characteristics of rabbit muscle pyruvate kinase (PK), a tetrameric enzyme having identical subunits, were investigated unde r neutral as well as acidic conditions by using enzymatic activity measurem ents and a combination of optical methods, such as circular dichroism, fluo rescence, and ANS binding. At low pH and low ionic strength, pyruvate kinas e exists in a partially unfolded state (U-A state) retaining half of the se condary structure and no tertiary interactions along with a strong binding to the hydrophobic dye, ANS. Addition of anions, like NaCl, KCl, and Na2SO4 , to the acid-unfolded state induces refolding, resulting structural propen sities similar to that of native tetramer. When anion concentration exceeds a critical limit (0.7 M KCl), a sudden loss of secondary structure and dec rease in fluorescence intensity with a redshift in the emission maximum are seen which may be due to the aggregation of the protein, probably due to t he intermolecular association. The anion-refolded state is more stable than . the U-A state, and its stability is nearly equal to that of native protei n toward chemical-induced unfolding by Gu-HCl and urea. Moreover, at low co ncentrations, Gu-HCl behaves like an anion, by inducing refolding of the ac id-unfolded state with structural features equivalent to that of native mol ecule. These observations support a model of protein folding where certain conformations of low free energy prevail and are populated under non-native conditions with different stability. (C) 2000 Academic Press.