A Tour of Carbonic Anhydrase

Please be patient while the structures in the left frame load. In order to display all of the structures in the tour properly, press 'View' buttons below in order (from 1 to the end).

Crystals of carbonic anhydrase II

The crystals of the bacterial carbonic anhydrase   [1]

Carbonic anhydrase is a hydrolytic enzyme that catalyzes the addition of water to carbon dioxide:

CO2 + H2O     HOCO2- + H+

The rate of catalysis is very pH dependent, faster at higher pH, with pH=6.5 being the optimum rate. This enzyme is found in the red blood cells of humans along with hemoglobin.

There are three broad classes of carbonic anhydrases:

Regardless of the class to which they belong, all carbonic anhydrases perform the same function and have the same active site. The difference lies in the amino acid sequence and the catalytic efficiency.

The other interesting feature of this enzyme is the existence of a large number of isoenzymes (or isozymes)[2]. For example at least 14 isozymes of the α class carbonic anhydrases have been identified. Some of them are the most efficient catalysts known (the rate constant of the catalysed reaction is 107 times faster than a non-catalysed reaction).

The wireframe display in the left screen shows the structure of the bovine carbonic anhydrase II, one of its seven (I to VII) isozymes. It contains 259 amino acid residues with a molecular weight of 29 kDa.

  The six α-helices (red) and 10 β-sheets (blue) make up the secondary structure of this monomeric enzyme.

  Through the protein structure runs a channel with a diameter of around 15 . The active site, containing one zinc(II) ion, resides inside this channel.

  The zinc(II) cation is connected to the protein backbone via three histidine residues: His93, His95 and His118. These residues are located on the anti-parallel β-sheets connected by a hydrogen bond network and are on the same side of the sheet. This conformation gives a tridentate ligand.

  In this display the protein backbone has been removed and only the active site is shown. The fourth ligand on the Zn(II) ion is a water molecule. The zinc coordination is tetrahedral. The three histidine residues are sometimes referred to as first coordination sphere ligands. This set of ligands is appropriate to give the water ligand the pKa of 6.5, a value where catalysis is most efficient. The aqua ligands in [Zn(OH2)6]2+ have a pKa of 9.6, so three histidines delocalize less positive charge from the Zn(II) than 5 aqua (water) ligands. Every hydrogen on each aqua ligand stabilizes some positive charge. Click to see the coordination polyhedron.

  This display shows the amino acid residues from the second coordination sphere. One of their functions is to stabilize the histidine ligands thrugh hydrogen bonding: His93 to Gln91 and His118 to Glu116. By forming these H-bonds, these residues also help position the histidine ligands for the coordination with zinc.

  The second function of the residues in the second sphere is to form a hydrogen bond network with a number of water molecules (red spheres) located around the active site. This network provides a path required for proton transfer.

This display shows two amino acids from the second coordination sphere that play an additional role. The His63 acts as a base responsible for the deprotonation of the water molecule coordinated to the Zn(II). The deprotonation is not direct, but rather goes via the hydrogen bond network shown above. The Thr197 is hydrogen bonded to the OH- on the zinc ion. This interaction helps orient the hydroxyl ion for the optimal nucleophilic attack on the CO2. It is said that this amino acid serves as a "gatekeeper" because, through hydrogen bonds, it allows only protonated substrates to interact with the zinc cation.

For the carbonic anhydrase catalytic cycle click here.

This is a display of carbonic anhydrase to explore further. Feel free to play around with it and all the other molecules displayed in the other pages.

The structure of carbonic anhydrase by Saito, R.; Sato, T.; Ikai, A.; Tanaka, N. (2004) Acta Cryst. D 60, 792-795 (PDB id 1V9E).

Carbonic Anhydrase - A Model Complex

  The structure on the left side represents a synthetic model of the active site of carbonic anhydrase having three imidazolyl ligands and a OH- group coordinated to Zn2+[3]. A common problem in the synthesis of similar compounds is the formation of bimetallic species bridged by the OH groups. This is avoided here by placing bulky t-butyl substituents on the imidazolyl rings. These sterically demanding groups prevent the dimerization.

  This is another interesting model of the carbonic anhydrase active site, [HB(3-tBu-5-Me-pz)3]Zn(OH), [HB(3-tBu-5-Me-pz)3 = tris(3-ter-butyl-5-methyl-pyrazolyl)hydroborate][4].The coordination geometry around the zinc ion is tetrahedral with a trigonal distortion as indicated by the three small N-Zn-N bond angles [92.8(2)o] and the three large N-Zn-O angles [123.2(2)o]. The solutions of this complex can absorb CO2 giving a new compound that has been identified as a bicarbonate complex, [HB(3-tBu-5-Me-pz)3]Zn(OCO2H) based on the IR spectroscopy. The hydroxyl complex can also be reversibly protonated to give the conjugate acid {[HB(3-tBu-5-Me-pz)3]Zn(OH2)}+ [5]. The solutions of the acid form are unable to react with carbon dioxide showing that the deprotonation of zinc-bound water is an essential step in the mechanism of carbonic anhydrase.

[1] Picture taken from Chemistry @ Colgate

[2] Izoenzyme (or isozyme) is any of the chemically distinct forms of an enzyme that perform the same biochemical function.

[3] Kimblin, C.; Allen, W.E.; Parkin, G. (1995) J. Chem. Soc., Chem. Commun. 1813.

[4] Alsfasser, R.; Trofimenko, S.; Looney, A.; Parkin, G.; Vahrenkamp, H. (1995) Inorg. Chem., 30(21), 4098.

[5] (a) Bergquist, C; Parkin, G. (1999) J. Am. Chem. Soc., 121(26), 6322. (b) Bergquist, C.; Fillebeen, T.; Morlok, M.M; Parkin, G. (2003) J. Am. Chem. Soc. , 125(20), 6189

Copyright Robert H. Morris, Adrian Lee and Alen Hadzovic, 1998, 2009,2011.

Creative Commons License
The Guided Tours of Metalloproteins by Alen Hadzovic and Robert H. Morris is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License