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Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
Published on: June 30, 2019
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Structural Investigation of a Dimeric Variant of Pyruvate Kinase Muscle Isoform 2.
Dhiraj Srivastava1, Mortezaali Razzaghi1, Michael T Henzl2
1Department of Chemistry, University of Iowa , Iowa City, Iowa 52242, United States.
Biochemistry
|November 29, 2017
Summary
The S437Y variant of pyruvate kinase muscle isoform 2 (PKM2) cannot bind fructose 1,6-bisphosphate (FBP). This impairs its enzymatic activity, revealing insights into PKM2
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Pyruvate kinase muscle isoform 2 (PKM2) is a key glycolytic enzyme.
- PKM2 activity is regulated by fructose 1,6-bisphosphate (FBP), which stabilizes its active tetrameric form.
- FBP binding is crucial for maintaining high PKM2 enzymatic activity.
Purpose of the Study:
- To investigate the impact of the S437Y mutation on PKM2 structure and function.
- To elucidate the structural basis for impaired FBP binding in the S437Y PKM2 variant.
Main Methods:
- Biochemical assays to determine enzyme kinetics and binding affinity.
- Analytical ultracentrifugation to study protein oligomerization states.
- X-ray crystallography to determine the structure of the S437Y PKM2 variant.
Main Results:
- The S437Y PKM2 variant exhibits impaired FBP binding.
- S437Y PKM2 exists in a monomer-dimer equilibrium in solution (Kd ≈ 20 μM).
- Despite solution behavior, S437Y PKM2 crystallizes as a tetramer, offering structural insights.
Conclusions:
- The S437Y mutation disrupts FBP-mediated stabilization of the PKM2 tetramer.
- Structural analysis of the S437Y variant provides a molecular understanding of FBP binding.
- This study deepens our knowledge of PKM2 allosteric regulation and its implications in glycolysis.
