Efficient proton shuttle makes SazCA an excellent CO₂ hydration enzyme

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Carbonic anhydrases are crucial enzymes catalyzing CO2 hydration.
• SazCA is the fastest known carbonic anhydrase, showing high activity at elevated temperatures (353 K).
• Understanding SazCA's molecular basis for high activity is essential for enzyme engineering.

Purpose of the Study:

• To elucidate the molecular mechanisms behind SazCA's exceptional catalytic activity.
• To compare SazCA with a related enzyme, SspCA, using molecular dynamics simulations.
• To identify key residues and conformational states contributing to SazCA's enhanced function.

Main Methods:

• Molecular dynamics (MD) simulations at various temperatures.
• Analysis of proton shuttle efficiency between the active site and His64.
• Root-mean-square fluctuation (RMSF) and hydrogen bond (H-bonds) analysis.

Main Results:

• Both SazCA and SspCA possess efficient proton shuttle systems involving His64.
• His64 adopts 'in' and 'out' conformations, with 'in' favoring proton acceptance.
• SazCA exhibits a significantly higher population of the 'in' conformation compared to SspCA.
• His2 and His207 in SazCA play a role in stabilizing the 'in' conformation of His64.

Conclusions:

• The superior activity of SazCA is attributed to a greater prevalence of the proton-accepting 'in' conformation of His64.
• Specific residues (His2, His207) in SazCA contribute to stabilizing this active conformation.
• These findings provide insights into the structural basis of high enzyme activity in carbonic anhydrases.