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 investigate the homotropic and heterotropic cooperative interactions of the enzyme. Cooperative ligand binding by the wild-type enzyme was measured using pre-steady-state and steady-state fluorescence spectroscopy, and steady-state kinetics. The results suggest that the cooperative structural changes induced by the substrate phosphoenolpyruvate (PEP) are distinct from those induced by the allosteric activator ribose- 5-phosphate (R5P). Furthermore the structural transition induced by the binding of saturating amounts of both PEP and R5P is itself distinct. This conclusion was further substantiated by the production of five mutant proteins in which the R5P- and PEP-induced homotropic cooperative transitions were separated. These results suggest that the cooperativity exhibited by pyruvate kinase from B. stearothermophilus does not conform to a simple two-state model. A putative four-state model is proposed.
Lovell
S C
SC
Department of Biochemistry and Molecular Recognition Centre, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
Mullick
A H
AH
Muirhead
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012267:Ribosemonophosphates / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D008024:Ligands; D012267:Ribosemonophosphates; C031626:ribose-5-phosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase