Structural and kinetic effects on changes in the CO(2) binding pocket of human carbonic anhydrase II

Abstract

This work examines the effect of perturbing the position of bound CO(2) in the active site of human carbonic anhydrase II (HCA II) on catalysis. Variants of HCA II in which Val143 was replaced with hydrophobic residues Ile, Leu, and Ala were examined. The efficiency of catalysis in the hydration of CO(2) for these variants was characterized by (18)O exchange mass spectrometry, and their structures were determined by X-ray crystallography at 1.7-1.5 Å resolution. The most hydrophobic substitutions, V143I and V143L, showed decreases in the level of catalysis, as much as 20-fold, while the replacement by the smaller V143A mutation showed an only moderate 2-fold decrease in activity. Structural data for all three variants show no significant change in the overall position of amino acid side chains in the active site compared with the wild type. However, V143A HCA II showed additional ordered water molecules in the active site compared to the number for the wild type. To further investigate the decrease in the catalytic efficiency of V143I HCA II, an X-ray crystallographic CO(2) entrapment experiment was performed to 0.93 Å resolution. This structure revealed an unexpected shift in the CO(2) substrate toward the zinc-bound solvent, placing it ~0.3 Ǻ closer than previously observed in the wild type in conjunction with the observed dual occupancy of the product bicarbonate, presumably formed during the acquisition of data. These data suggest that the Ile substitution at position 143 reduced the catalytic efficiency, which is likely due to steric crowding resulting in destabilization of the transition state for conversion of CO(2) into bicarbonate and a decreased product dissociation rate.

Figure 1

Stick diagram of the active site of HCA II. Substrate CO₂ (orange), active site solvent (red spheres), hydrophobic pocket (light gray), the hydrophilic region (blue), and the three histidines (yellow) stabilizing the zinc (dark gray). PDB ID: 2ILI (10). Amino acids are as labeled. This figure was generated with PyMol (32).

Figure 2

pH profiles of k_cat^ex/K_eff^CO2 (μM⁻¹ s⁻¹) for the hydration of CO₂ catalyzed by variants of HCA II: wild type (black, ●); V143A (blue, △); V143I (red, ■); and V143L (green, ◇). Data were obtained from rates of depletion of ¹⁸O from CO₂ measured by membrane inlet mass spectrometry at 25 °C in solutions containing 25 mM ¹⁸O-enriched CO₂/bicarbonate. No buffers were added.

Figure 3

Crystal structure at the active-site of the A) V143A (pink), B) V143L (yellow), and C) V143I HCAII (orange). Amino acids are as labeled. Shown are the calculated 2F_o-F_c electron density map for the 143 position, contoured at 1.5σ (blue). Solvent molecules are depicted as red spheres and the three histidines coordinating the zinc (dark gray). This figure was generated with PyMol (32).

Figure 4

Stick overlay of position 143 HCA II structures. Wild type (orange), V143A (pink), V143L (yellow), and V143I HCAII (white). Amino acids are as labeled. In addition the three histidines coordinating the zinc (dark gray) are shown. This figure was generated with PyMol (32).

Figure 5

Stick stereo figure of the active site of V143I HCA II CO₂/bicarbonate complex. Shown are the calculated omit Fo-Fc electron density map, contoured at 3σ (green), when the A) CO₂ and B) bicarbonate were not incorporated into the respective models. The residual electron density in either case indicates presence of both substrate and product. Amino acids are as labeled. This figure was generated with PyMol. (32).

Figure 6

Structure of substrate/product bound in HCAII V143I. A) CO₂ and H₂O molecules (red sphere), B) HCO₃⁻, and C) as observed in the crystal structure, with dual occupancy (Refer to Figure 5). Amino acids are as labeled. This figure was generated with PyMol (32).

Figure 7

Comparison of HCA II V143I substrate and product binding in HCA II. Structural overlay A) of CO₂ binding in wild type (green) (6) and V143I (yellow) HCAII, B) of bicarbonate binding in T200H (green) (21) and V143I (yellow). Amino acids are as labeled. This figure was generated with PyMol (32).