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Electron spin echo envelope modulation studies of pyruvate kinase active-site complexes
P A Tipton1, J McCracken, J B Cornelius
1Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Biochemistry
|July 11, 1989
Summary
Electron spin echo envelope modulation (ESEEM) spectroscopy revealed distinct active-site structures in pyruvate kinase. It showed how substrates and cofactors influence metal ion coordination, clarifying enzyme mechanisms.
Area of Science:
- Biochemistry
- Enzymology
- Spectroscopy
Background:
- Pyruvate kinase is a key enzyme in glycolysis.
- Understanding its active-site structure is crucial for enzyme mechanism elucidation.
- Divalent metal ions play critical roles in enzyme catalysis.
Purpose of the Study:
- To investigate the active-site structure of rabbit muscle pyruvate kinase.
- To examine the coordination of metal ions (Mn2+, VO2+) with substrates and cofactors.
- To elucidate the role of monovalent cations in enzyme-metal interactions.
Main Methods:
- Electron spin echo envelope modulation (ESEEM) spectroscopy.
- Utilized Mn2+ and VO2+ as paramagnetic probes.
- Studied interactions with substrates (pyruvate, PEP), products (MgATP), and inorganic cofactors (Na+, K+, Cs+).
Main Results:
- Distinguished two VO2+-protein complexes based on lysine coordination, sensitive to substrates and monovalent cations.
- Observed direct ATP coordination to Mn2+ via 31P contact interaction in the presence of MgATP and oxalate.
- Determined pyruvate as a bidentate ligand to VO2+ using 13C ESEEM.
- Demonstrated proximity of monovalent cations (Na+, K+, Cs+) to the protein-based divalent cation site.
Conclusions:
- ESEEM spectroscopy effectively probes enzyme active-site structures.
- Metal ion coordination and substrate binding are dynamic and influenced by reaction conditions.
- This study provides detailed insights into the structural basis of pyruvate kinase function.