Entrapment of carbon dioxide in the active site of carbonic anhydrase II

Abstract

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO(2)-pressurized, cryo-cooled crystals to capture the first step of CO(2) hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO(2), for both the holo and the apo (without zinc) enzyme to 1.1A resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO(2) and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO(2) in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO(2). This method may also have a much broader implication for the study of other enzymes for which CO(2) is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

FIGURE 1.

hCAII structure. a, overall view, showing the hydrophilic (magenta stick representation) and hydrophobic (green surface representation) sides of the active site. The active site zinc is shown in purple with the waters of the proton wire shown as small, red spheres. b and c, a close-up stereo view of the active site showing the position of bound CO₂ in holo- (b) and apohCAII (c). Electron density of the active site amino acids and W_I (σ-weighted 2F_o - F_c Fourier map contoured at 2.25 σ) and CO₂ (σ-weighted F_o - F_c Fourier map contoured at 2.25 σ). The figure was created using PyMOL.

FIGURE 2.

Second CO₂ binding site. a, surface representation showing the separation of the active site (green) and non-catalytic (pink) CO₂ binding pockets. b, close-up view of the CO₂ binding. Note the conformational change in Phe-226 (red = unbound, green = CO₂-bound holohCAII). The electron density is a σ-weighted 2F_o - F_c Fourier map contoured at 1.5 σ. The figure was created using PyMOL.

FIGURE 3.

Glycerol binding sites in CO₂-bound hCAII structures. a, ordered glycerol molecule located at mouth of the active site in the holo enzyme. b and c, second ordered glycerol observed bound on the surface of the holo (b) and apo (c) structures. The electron density maps are 2F_o - F_c Fourier map contoured at 1.0 σ. The figure was made using PyMOL.

FIGURE 4.

Active site. a, superposition of unbound holo- (13), CO₂-bound holo-, and bicarbonate-bound T200H hCAII (32). The binding modes of both the CO₂ substrate and the product are similar, with the substrate favoring the hydrophobic side (green) and product favoring the hydrophilic side (orange). Note that the deep water (W_DW, gray sphere) is displaced and a new water occupies the area between the side chain of Thr-200 and CO₂ (W_I, cyan) upon CO₂ binding. The CO₂ is orientated so that the carbon is primed for the nucleophilic attack by the zinc-bound hydroxide (orange sphere). b, a superposition of the V143Y variant of hCAII (9, 27). Note that the side chain of Tyr-143 (white) acts as a steric block to the CO₂ binding site. The figure was created using PyMOL.

FIGURE 5.

Proposed mechanisms of hCAII catalysis. a and b, Lipscomb (32) mechanism (a) and Lindskog (33) (b) mechanism.

FIGURE 6.

Proposed catalytic mechanism of hCAII. A schematic representation of three discrete stages of the catalytic cycle is shown. a, unbound. Note the presence of deep water (W_DW). b, CO₂ bound. Note the displacement of W_DW and the hydrogen bond between the substrate and backbone amide of Thr-199. c, the formation of bicarbonate. The figure was created using ChemDraw 11.0 (available from CambridgeSoft).