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Plant cytosolic pyruvate kinase: a kinetic study
1Department of Biology, Queen's University, Kingston, Ontario, Canada.
Biochimica Et Biophysica Acta
|November 20, 1992
Summary
This study reveals that cytosolic pyruvate kinase (PKc) in castor oil seeds uses free phosphoenolpyruvate (PEP) and ADP as substrates. The enzyme naturally exists in an activated state, influencing glycolytic flux.
Area of Science:
- Biochemistry
- Enzymology
- Plant Physiology
Background:
- Cytosolic pyruvate kinase (PKc) plays a crucial role in glycolysis.
- Understanding PKc kinetics is vital for comprehending plant metabolic regulation.
- Castor oil seeds (COS) present a unique system for studying plant PKc.
Purpose of the Study:
- To investigate the kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS).
- To determine the true substrates and reaction mechanism of COS PKc.
- To elucidate the regulatory state and implications for glycolytic flux in plants.
Main Methods:
- Enzyme kinetics experiments varying free Mg2+ and substrate concentrations.
- Intrinsic tryptophan fluorescence quenching assays with substrates.
- Analysis of product and analogue inhibition patterns.
Main Results:
- Free phosphoenolpyruvate (PEP) and ADP were identified as the true substrates for COS PKc.
- A sequential, compulsory-ordered Tri-Bi kinetic mechanism was proposed, with PEP as the leading substrate.
- COS PKc exhibits kinetic properties indicating a naturally activated state, with lower substrate affinities than non-plant enzymes.
Conclusions:
- COS PKc operates in an intrinsically activated state, independent of allosteric activators.
- The enzyme's regulation is likely influenced by ATP levels and pH, affecting its response to inhibitors.
- Regulation of PKc by varying PEP levels is a key determinant of glycolytic flux in the plant cytosol.