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Cooperativity in Bacillus stearothermophilus pyruvate kinase
S C Lovell1, A H Mullick, H Muirhead
1Department of Biochemistry and Molecular Recognition Centre, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, U.K.
Journal of Molecular Biology
|May 9, 1998
Summary
Bacillus stearothermophilus pyruvate kinase (PK) exhibits complex cooperativity. Ligand binding induces distinct structural changes, suggesting a four-state model rather than a simple two-state mechanism for this enzyme.
Area of Science:
- Enzymology
- Protein dynamics
- Biophysical chemistry
Background:
- Pyruvate kinase (PK) is a key glycolytic enzyme.
- Understanding enzyme cooperativity is crucial for metabolic regulation.
- Bacillus stearothermophilus PK serves as a model for studying enzyme mechanisms.
Purpose of the Study:
- To investigate the homotropic and heterotropic cooperative interactions of Bacillus stearothermophilus pyruvate kinase (PK).
- To elucidate the distinct structural changes induced by substrate (phosphoenolpyruvate, PEP) and allosteric activator (ribose-5-phosphate, R5P) binding.
- To determine if PK cooperativity fits a simple two-state model.
Main Methods:
- Pre-steady-state and steady-state fluorescence spectroscopy.
- Steady-state kinetics.
- Site-directed mutagenesis to produce five mutant proteins.
Main Results:
- Cooperative ligand binding by wild-type B. stearothermophilus PK was observed.
- Structural changes induced by PEP binding are distinct from those induced by R5P binding.
- A distinct structural transition occurs upon simultaneous binding of PEP and R5P.
- Mutant proteins demonstrated separation of R5P- and PEP-induced cooperative transitions.
Conclusions:
- The cooperativity of B. stearothermophilus PK does not conform to a simple two-state model.
- A more complex, putative four-state model is proposed to explain the observed ligand binding and structural changes.
- Distinct conformational states are involved in substrate and allosteric activator binding to PK.