Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II

Abstract

Bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge, as several stabilizing elements have been proposed to be the foundation of thermal stability, including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions and aromatic clusters. This study emphasizes the effect of increasing the rigidity of human carbonic anhydrase II (HCA II; EC 4.2.1.1) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of the compactness of HCA II was examined by deletion of a surface loop (residues 230-240) that had been previously identified as a possible source of thermal stability for the hyperthermophilic carbonic anhydrase isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme, while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed that the electrostatic potential and configuration of the highly acidic loop (residues 230-240) play an important role in its high catalytic activity. Based on these observations and previous studies, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements may provide blueprints for rational thermal stability engineering of other enzymes.

Database: Structural data have been submitted to the Protein Data Bank under accession numbers 4QK1 (K170P), 4QK2 (E234P) and 4QK3 (Δ230-240).

Keywords: bioengineering; carbonic anhydrase; differential scanning calorimetry; protein crystallography; protein thermal stability.

Figure 1

Thermograms of the unfolding transition for A) wild-type; B) K170P; C) E234P; and D) Δ230–240 HCA II at pH 7.8. The buffer subtracted, baseline-corrected data are shown in black with the fit to the data shown in red.

Figure 2

pH profiles for A) catalytic efficiency (k_cat/K_M) and B) proton transfer rate (R_H2O/[E]) for the HCA II variants. Wild-type is shown as a closed square, K170P as an open diamond, E234 as an open triangle and Δ230–240 HCA II as a closed circle.

Figure 3

Topology of the 230–240 loop in the HCA II variants. A) Cartoon view of wild-type HCA II (green) with the 230–240 loop highlighted in purple. The Zn²⁺ (magenta sphere) with the coordinating His residues and the proton shuttle residue, His64 shown in stick. B) Comparison of the 230–240 loop in the K170P (blue) to wild-type (green) HCA II. Side-chains are shown in stick and are as labeled. Residues G233 and G235 are not labeled for clarity. C) Comparison of the 230–240 loop in E234P (light purple) to wild-type (green) HCA II. Residues are as labeled with potential salt bridge interactions shown as a dashed line. D) Ligation between residues L229 and M241 in Δ230–240 (light pink) showing the deletion of the loop compared to wild-type HCA II (green) HCA II.

Figure 4

Active site water network configuration in Δ230–240 (red spheres) overlaid with the wild-type (light pink spheres) HCA II. Potential hydrogen bond interactions for Δ230–240 are shown as black dashed lines whereas the white dashed lines represent the hydrogen bonds observed in wild-type HCA II Residues and water molecules are as labeled.

Figure 5

Schematic of the proton transfer network for A) wild-type and B) Δ230–240 HCA II. Hydrogen bond interactions (not drawn to scale) are depicted as arrows with the distance between atoms label in Å.