Common enzymological experiments allow free energy profile determination.

Area of Science:

• Biochemistry
• Enzymology
• Chemical Kinetics

Background:

• Determining complete sets of rate constants, or free energy profiles (FEPs), for complex enzymatic mechanisms is difficult.
• Enzymologists use various steady-state kinetic experiments (e.g., Michaelis-Menten kinetics, kinetic isotope effects, viscosity dependence) to gain insights into kinetic systems.

Purpose of the Study:

• To present a straightforward method for integrating diverse steady-state kinetic experiments into a single analysis.
• To enable the determination of microscopic rate constants and intrinsic kinetic isotope effects.

Main Methods:

• Utilizing Michaelis-Menten parameters (kcat, Km), kinetic isotope effects, solvent viscosity effects, and intermediate partitioning measurements.
• Applying global optimization to integrate independent experimental data.
• Employing algorithms within the COPASI software for rate constant determination.

Main Results:

• Demonstrated the sufficiency of combined steady-state experiments to define rate constants for a reversible uni-uni mechanism with an intermediate.
• Successfully applied the method to determine FEPs for alanine racemase and triosephosphate isomerase.
• Obtained FEPs that largely agree with existing literature values, with notable discrepancies.

Conclusions:

• The developed method offers a simplified approach for combining multiple steady-state kinetic experiments.
• This technique facilitates the routine and large-scale determination of enzyme free energy profiles.
• The findings provide a valuable tool for advancing our understanding of enzyme kinetics.