Bacillus subtilis chorismate mutase is partially diffusion-controlled

Area of Science:

• Biochemistry
• Enzymology
• Protein Dynamics

Background:

• Chorismate mutase catalyzes a key reaction in aromatic amino acid biosynthesis.
• Understanding enzyme kinetics and reaction mechanisms is crucial for metabolic pathway analysis.
• Viscosity can probe the role of diffusion in enzyme-catalyzed reactions.

Purpose of the Study:

• To investigate the impact of microviscosity on the catalytic activity of Bacillus subtilis chorismate mutase (BsCM).
• To elucidate the rate-limiting steps in the BsCM-catalyzed rearrangement of chorismate to prephenate.
• To differentiate between diffusion-limited steps and chemical steps in enzyme catalysis.

Main Methods:

• Enzyme kinetics assays using varying concentrations of viscosogens (e.g., glycerol).
• Characterization of wild-type BsCM and specific mutants (C75A, C75S) under different viscosity conditions.
• Analysis of steady-state kinetic parameters (kcat, kcat/Km) and substrate/product 'on' rates.

Main Results:

• Wild-type BsCM activity decreased significantly with increasing glycerol concentration, indicating viscosity dependence.
• BsCM mutants C75A and C75S were insensitive to microviscosity changes, ruling out non-specific viscosogen interactions.
• Kinetic data suggest diffusion contributes 30-40% to the rate-determining steps, involving substrate binding and product release.

Conclusions:

• The catalytic mechanism of BsCM is influenced by microviscosity, primarily affecting substrate binding and product release.
• Diffusion of substrates and products plays a significant role in the overall reaction rate.
• The accessibility of the active site influences viscosity sensitivity, as seen in the comparison with E. coli chorismate mutase.