Nonconserved active site residues modulate CheY autophosphorylation kinetics and phosphodonor preference

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Two-component signal transduction relies on response regulators for cellular signaling.
• Response regulators can autophosphorylate, a crucial process for signal transduction and experimental manipulation.
• The kinetic factors governing response regulator autophosphorylation remain largely uncharacterized.

Purpose of the Study:

• To investigate the autophosphorylation kinetics of Escherichia coli CheY variants.
• To identify the kinetic determinants influencing autophosphorylation rates.
• To explore the impact of active site residue substitutions on phosphodonor preference and reaction rates.

Main Methods:

• Characterization of autophosphorylation kinetics for 21 CheY variants with substitutions at D+2 and T+2 positions.
• Assays using phosphoramidate, acetyl phosphate, and monophosphoimidazole as phosphodonors.
• Analysis of rate constants (kphos/KS) and their dependence on ionic strength and side chain properties.

Main Results:

• CheY variants displayed a >10^5-fold range in autophosphorylation rate constants.
• Most variants showed enhanced rates compared to wild-type CheY, with phosphoramidate often being the preferred donor.
• Increased positive charge and nonpolar surface area of active site side chains correlated with faster autophosphorylation rates.

Conclusions:

• Electrostatic and nonpolar interactions at active site positions D+2 and T+2 are critical determinants of CheY autophosphorylation kinetics.
• Ionic strength sensitivity highlights the role of both long-range and localized electrostatic interactions.
• Structural mimicry may explain accelerated rates in certain variants, suggesting implications for phosphotransfer mechanisms.