Modulation of Response Regulator CheY Reaction Kinetics by Two Variable Residues That Affect Conformation

Area of Science:

• Microbiology and Plant Science
• Biochemistry
• Molecular Biology

Background:

• Microorganisms and plants use two-component systems for environmental adaptation.
• Sensor kinases and response regulators mediate signal transduction through phosphorylation.
• Previous work identified D+2 and T+1/T+2 positions affecting response regulator kinetics.

Purpose of the Study:

• To investigate the kinetic effects of amino acid substitutions at the K+1/K+2 positions in the Escherichia coli response regulator CheY.
• To determine how these substitutions influence autophosphorylation and autodephosphorylation rates.
• To elucidate the mechanism by which K+1/K+2 positions modulate reaction kinetics.

Main Methods:

• Measured autophosphorylation and autodephosphorylation rate constants for 27 pairs of K+1/K+2 residues in CheY.
• Analyzed the correlation between autophosphorylation rates and dissociation constants for a phosphoryl group analog (BeF3-).
• Assessed the independence of kinetic effects from the phosphodonor.

Main Results:

• Autophosphorylation rate constants varied by two orders of magnitude across K+1/K+2 substitutions.
• Autodephosphorylation effects were modest.
• Kinetic modulation appears to be indirect, affecting CheY's conformational equilibrium rather than directly interacting with the phosphorylation site.

Conclusions:

• The K+1/K+2 positions significantly influence CheY autophosphorylation kinetics, likely by altering protein conformation.
• Five variable positions in response regulators allow for a three-orders-of-magnitude tuning of reaction rates.
• This understanding can inform synthetic biology applications for biosensor development and kinetic prediction.