Rigidifying a De Novo Enzyme Increases Activity and Induces a Negative Activation Heat Capacity

Area of Science:

• Enzyme kinetics and biocatalysis
• Protein conformational dynamics
• Biophysical chemistry

Background:

• Peroxidases are valuable biocatalysts for various transformations.
• Enzyme activity and temperature dependence are significantly influenced by conformational sampling.
• De novo enzymes offer potential for tailored biocatalytic applications.

Purpose of the Study:

• To investigate the role of conformational sampling in the activity of the de novo peroxidase C45.
• To understand how 2,2,2-trifluoroethanol (TFE) affects enzyme structure, stability, and activity.
• To compare the catalytic thermodynamics of the engineered enzyme with a natural peroxidase.

Main Methods:

• Enzyme activity assays across a range of temperatures.
• Use of 2,2,2-trifluoroethanol (TFE) to modulate enzyme conformation.
• Application of macromolecular rate theory (MMRT) to analyze temperature dependence data.
• Thermodynamic analysis of enzyme catalysis.

Main Results:

• TFE treatment induced a more rigid conformational state in C45, enhancing stability and activity.
• The temperature-activity profile of TFE-treated C45 exhibited curvature, indicating a trade-off between low and high-temperature performance.
• MMRT analysis suggested increased protein rigidity and altered dynamics between ground and transition states.
• The modified C45 showed thermodynamic and dynamic properties similar to horseradish peroxidase.

Conclusions:

• Manipulating protein rigidity offers a generic strategy to enhance biocatalytic activity.
• TFE can induce enzyme-like behavior in de novo catalysts, improving their performance without extensive engineering.
• This study provides insights into optimizing de novo enzymes for industrial applications.