Utilization of conformational flexibility in enzyme action-linkage between binding, isomerization, and catalysis

Area of Science:

• Biochemistry
• Enzyme kinetics
• Structural biology

Background:

• A close link between protein conformational changes and enzyme catalysis is frequently proposed.
• Understanding this relationship is crucial for enzyme mechanism elucidation.

Purpose of the Study:

• To investigate the connection between ligand binding, protein conformational changes, and reaction rates in Escherichia coli ornithine transcarbamoylase.
• To determine if substrate-induced isomerization is a key step in the enzyme's catalytic mechanism.

Main Methods:

• Utilized ligand-induced ultraviolet difference spectroscopy to monitor protein conformational changes.
• Determined kinetic parameters for wild-type and site-specific mutants of ornithine transcarbamoylase.
• Analyzed the correlation between binding energy, activation energy, and spectral changes.

Main Results:

• Carbamoyl phosphate binding induced significant conformational changes in wild-type ornithine transcarbamoylase, evidenced by UV difference spectra and crystal cracking.
• Mutants showed similar spectral lineshapes but varying intensities, correlating with altered substrate affinity and kinetic parameters.
• A strong inverse correlation was observed between the magnitude of substrate-induced spectral changes and the calculated binding/activation energies.

Conclusions:

• Substrate binding to ornithine transcarbamoylase triggers a committed isomerization essential for transition state binding.
• This induced-fit isomerization plays a significant role in controlling the enzyme's catalytic rate.
• Provides direct evidence for the functional importance of ligand-induced conformational dynamics in enzyme catalysis.