Structural Characterization of Mutant Carbonic Anhydrase via High-Pressure Cryogenic X-ray Crystallography

Abstract

Carbonic anhydrase II (CAII) is a zinc-containing metalloenzyme that catalyzes the reversible hydration of carbon dioxide, a reaction critical for physiological pH regulation, respiration, and ion transport. The catalytic efficiency of CAII (kcat/KM ~ 10^8 M^-1^s-1) is underpinned by an intricately organized network of active-site water molecules which together facilitate rapid proton transfer and substrate turnover. Among active-site residues, Thr200 plays a pivotal role by hydrogen-bonding to key water molecules (W1, WI, WI′), anchoring the proton-transfer “wire” and replenishment pathway that connect the Zn-bound hydroxide to bulk solvent. Substitution of Thr200 with bulky histidine (T200H) is hypothesized to perturb these water networks, modulate proton-transfer kinetics, and thereby alter catalytic performance. In this study, high-pressure cryogenic X-ray crystallography is employed to trap and visualize intermediate states of wildtype and T200H CAII under defined CO2 atmospheres (0, 5, and 20 atm). Crystals were pressurized with CO2 and sequentially cryocooled at 100 K for diffraction data collection. The crystallographic snapshots captured the resting (T200H-0atm), substrate-bound (T200H-20atm), and product-bound (T200H-5atm) states of the T200H mutant. In the resting state, His200 disrupts the active site by displacing essential water molecules (W1 and W2), thereby impairing the proton transfer pathway. However, the substrate- and product-bound states reveal that His200 exhibits conformational flexibility, allowing partial restoration of the water network required for catalysis. These findings suggest that His200 functions as a dynamic gatekeeper, modulating access of water, substrate, and product to the active site. This structural plasticity explains how the T200H mutant retains partial catalytic activity despite a mutation that would otherwise severely hinder function. These structural perturbations correlate with steady-state kinetic measurements (kcat and KM) obtained via stopped‐flow spectroscopic assays using m-cresol purple indicator, which demonstrate a ~3-fold decrease in catalytic efficiency and altered pH–activity profiles for T200H CAII relative to wildtype. Together, this work includes theoretical background—including basic knowledge of carbonic anhydrase, X-ray crystallography, and high pressure cryocooling (HPC) method—and experimental methods such as expression and purification of protein and measurement of enzymatic activity. With the structural and functional results of T200H CAII, this work provides new insights into active-site dynamics in CAII and offer a foundation for designing isoform-specific inhibitors or engineered CA variants with tunable catalytic properties.