Mechanism of a Novel Metal-Free Carbonic Anhydrase

The recently discovered metal-free carbonic anhydrase (CA) enzyme may have a significant impact on the global carbon dioxide (CO2) cycle, as it can irreversibly perform the CO2 hydration reaction. In this study, we investigate several key aspects of metal-free CA, including the identification of the catalytic site, determination of the CO2 binding site, and the mechanism of catalysis. This is achieved through the use of 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 the metal-free CA, might not accurately depict the actual enzyme-substrate picture. Instead, the simulation reveals that the 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.