Transition-State Ensembles Navigate the Pathways of Enzyme Catalysis

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme kinetics
• Single-molecule biophysics

Background:

• Transition-state theory (TST) is crucial for understanding enzyme reaction rates.
• Existing TST models often overlook protein dynamics and catalytic heterogeneity.
• Enzyme catalysis involves complex interactions and conformational flexibility.

Purpose of the Study:

• To investigate enzyme reaction rate heterogeneities at the single-molecule level.
• To analyze the distribution of activation energy in enzyme ensembles.
• To elucidate the relationship between enzyme function, dynamics, and catalytic pathways.

Main Methods:

• Single-molecule enzyme kinetics analysis using large arrays of femtoliter chambers.
• Measurement of individual reaction rates for β-galactosidase and β-glucuronidase.
• Determination of the distribution of the free energy of activation (ΔG‡).

Main Results:

• Observed significant heterogeneities in individual enzyme reaction rates.
• β-galactosidase showed a broader distribution of ΔG‡ compared to β-glucuronidase.
• Attributed broader distribution in β-galactosidase to its dual hydrolase and transglycosylase functions.

Conclusions:

• Enzyme transition-state ensembles can direct catalytic pathways toward different products.
• β-galactosidase exemplifies evolutionary adaptation, with new pathways branching from existing functions.
• Functional specialization explains the larger conformational space of enzyme ensembles versus individual molecules.