COOPERATIVITY IN BACILLUS-STEAROTHERMOPHILUS PYRUVATE-KINASE

The enzyme pyruvate kinase (PK) from the moderate thermophile Bacillus stearothermophilus has been used as a model system with which to inve stigate the homotropic and heterotropic cooperative interactions of th e enzyme. Cooperative ligand binding by the wild-type enzyme was measu red using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative s tructural changes induced by the substrate phosphoenolpyruvate (PEP) a re distinct from those induced by the allosteric activator ribose-5-ph osphate (R5P). Furthermore the structural transition induced by the bi nding of saturating amounts of both PEP and R5P is itself distinct. Th is conclusion was further substantiated by the production of five muta nt proteins in which the R5P- and PEP-induced homotropic cooperative t ransitions were separated. These results suggest that the cooperativit y exhibited by pyruvate kinase from B. stearothermophilus does not con form to a simple two-state model. A putative four-state model is propo sed. (C) 1998 Academic Press Limited.