Memory landscapes of single-enzyme molecules

Area of Science:

• Biochemistry
• Enzyme kinetics
• Spectroscopy

Background:

• Horseradish peroxidase (HRP) is a crucial enzyme in various biological processes.
• Understanding enzyme dynamics at the single-molecule level is key to elucidating reaction mechanisms.
• Previous studies often analyze ensemble data, masking individual enzyme behaviors.

Purpose of the Study:

• To investigate the catalytic mechanism of single immobilized horseradish peroxidase enzymes.
• To explore the role of enzyme conformational dynamics in substrate turnover.
• To develop novel methods for analyzing single-enzyme kinetics and identifying transient states.

Main Methods:

• Confocal fluorescence spectroscopy was employed to monitor the enzymatic reaction in real-time.
• The non-Markovian behavior of the enzyme-product complex was extracted.
• Memory landscapes were generated from single-enzyme spectroscopic data.

Main Results:

• Single HRP enzymes catalyzed the conversion of dihydrorhodamine 6G to rhodamine 6G.
• Memory landscapes revealed oscillations, indicative of transient states within the enzyme.
• The analysis distinguished origins of stretched exponential kinetics observed in correlation analysis.
• Substrate interaction was shown to select active conformational substates.

Conclusions:

• Memory landscapes provide a powerful tool for analyzing single-enzyme dynamics and kinetics.
• Enzyme activity is modulated by substrate-induced selection of specific conformational substates.
• The study highlights the importance of transient states in enzyme catalysis.