PDBsum entry 3d93

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Lyase PDB id
Protein chain
257 a.a.
CO2 ×2
Waters ×352

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Title Entrapment of carbon dioxide in the active site of carbonic anhydrase ii.
Authors J.F.Domsic, B.S.Avvaru, C.U.Kim, S.M.Gruner, M.Agbandje-Mckenna, D.N.Silverman, R.Mckenna.
Ref. J Biol Chem, 2008, 283, 30766-30771. [DOI no: 10.1074/jbc.M805353200]
PubMed id 18768466
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 CO[2] 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 CO[2] (σ-weighted F[o] - F[c] Fourier map contoured at 2.25 σ). The figure was created using PyMOL.
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, CO[2] 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).
The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 30766-30771) copyright 2008.
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