Mechanism of a novel metal-free carbonic anhydrase

The recently discovered metal-free carbonic anhydrase (CA) enzyme may significantly impact the global carbon dioxide (CO₂) cycle, as it can irreversibly perform the CO₂ hydration reaction. In this study, we investigated several key aspects of metal-free CA, including the identification of the catalytic site, the determination of the CO₂ binding site, and the mechanism of catalysis. This is achieved through classical molecular dynamics (MD) simulations, quantum chemical density functional theory (DFT), and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. Our study indicates that the experimental structure based on X-ray crystallography, which shows the 'bicarbonate (HCO₃^-) product' trapped in the hydrophilic region of metal-free CA, might not accurately depict the actual enzyme-substrate interaction. Instead, the simulation reveals that CO₂ prefers the hydrophobic zone, which serves as the primary catalytic site. It also highlights the strategic role of a gatekeeper residue (Phe504), which assists in regulating the transportation of CO₂ by tilting its aromatic plane. Additionally, the hybrid QM/MM calculations establish that CO₂ hydration is catalyzed within the hydrophobic zone by a deprotonated tyrosine with the help of an organized water chain.