EXTRINSIC THERMOSTABILIZATION FACTORS AND THERMODENATURATION MECHANISMS FOR PHOSPHOENOLPYRUVATE CARBOXYLASE (PEPC) FROM AN EXTREMELY THERMOPHILIC BACTERIUM RHODOTHERMUS-OBAMENSIS

The thermodenaturation of phosphoenolpyruvate carboxylase (PEPC) from an extremely thermophilic bacterium Rhodothermus obamensis, capable of growth at temperature np to 85 degrees C, was probed at different den aturation temperatures by UV-visible absorption, fluorescence emission and 1-anilinonaphthalene-8-sulfonate (ANS) binding and renaturation w as assessed from different states of denaturation. Under severe denatu ration conditions at 100 degrees C, the enzyme was rapidly inactivated and its global structure immediately reached the irreversibly aggrega ted state by passing through the dissociated and the putative scramble d states as observed by UV-visible absorption spectroscopy. However, u nder milder conditions of denaturation at 93 degrees C, the enzyme was gradually inactivated, and its global structure shifted sequentially from the dissociated state to the scrambled state. At 80 degrees C, ab out 50% of the activity was left and no apparent change in the global structure occurred even after 30 h. In addition, ANS binding to the en zyme was greatly increased in accordance with the change in global str ucture. This implies that the hydrophobic regions of the enzyme tend t o be exposed to solvent due to thermal dissociation and unfolding. The extrinsic thermostabilization factors that enhance the thermostabilit y of the enzyme successfully suppress the thermodenaturation of the en zyme, especially the dissociation of its tetrameric form. Of these fac tors, the substrate for the enzyme, phosphoenolpyruvate (PEP), causes the reassociation of the dissociated inactive form of the enzyme to th e active form. These results suggest that the global thermodenaturatio n of the enzyme results from the temperature-dependent shift of three different states and that the extrinsic thermostabilization factors ac t to a large extent to maintain quaternary structure.