Interfacial communications in recombinant rabbit kidney pyruvate kinase

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Tissue-specific pyruvate kinase isozymes (muscle and kidney) exhibit distinct allosteric regulation despite originating from the same gene.
• Alternative mRNA splicing leads to minor sequence differences, causing significant variations in regulatory behavior.
• Understanding these differences is key to elucidating the molecular mechanisms of allostery.

Purpose of the Study:

• To clone and express rabbit kidney-type pyruvate kinase (rRKPK) in E. coli.
• To investigate the role of specific residues in conferring allosteric regulation.
• To explore the subunit assembly and its relation to allosteric effector binding.

Main Methods:

• Gene cloning and expression of rabbit kidney-type pyruvate kinase in Escherichia coli.
• Velocity sedimentation and analytical gel chromatography to study protein assembly.
• Analysis of dimer-tetramer equilibrium shifts upon substrate and effector binding.

Main Results:

• Recombinant rabbit kidney-type pyruvate kinase (rRKPK) and recombinant rabbit muscle-type pyruvate kinase (rRMPK) differ at 22 positions, primarily at intersubunit contact regions.
• rRKPK exhibits reversible dimer-tetramer assembly (equilibrium constant of 28 +/- 3 mL/mg).
• Substrate (PEP, ADP) and effector (FBP, Phe) binding influences the dimer-tetramer equilibrium, favoring tetramer formation with PEP and FBP, and dimer formation with ADP and Phe.

Conclusions:

• Allosteric substrates and effectors communicate through the dimer-dimer interface in pyruvate kinase.
• The thermodynamic responses to substrate and effector binding at this interface are distinct.
• This study provides new insights into the molecular mechanisms of allosteric regulation in pyruvate kinase systems.