CarbonicAnhydraseIIComplex

Acetazolamide-bound human carbonic anhydrase II (PDB ID: 6YMA) is an inhibited form of the enzyme that catalyzes the hydration of carbon dioxide to bicarbonate found in Homo sapiens. Free carbonic anhydrase function is best known in the bloodstream and in red blood cells. Carbonic anhydrase, when complexed with zinc, combines carbon dioxide with hydroxyl ions to form bicarbonate anions, which are water soluble (1). This is the mechanism through which erythrocytes in systemic capillaries release oxygen into the bloodstream. In the lungs, where carbon dioxide is secreted, the process is reversed. The native enzyme, without acetazolamide bound, has the same conformation as the acetazolamide-bound complex (2). Acetazolamide binds to human carbonic anhydrase II with high affinity and inhibits the enzyme activity (3). Facilitating the binding of acetazolamide to human carbonic anhydrase II has potential as a drug to treat glaucoma. Inhibiting carbonic anhydrase activity prevents the conversion of carbon dioxide and hydroxyl to bicarbonate, which in turn prevents the formation of the aqueous humor in the eye and lowering the intraocular pressure in glaucoma (2).

Human carbonic anhydrase II was expressed in BL21 DE3 pLysS E. Coli cells prior to crystallization. The protein was crystallized using sitting-drop vapor-diffusion set ups, with 2.8M ammonium sulfate and 0.1M Tris at pH 8.5 and temperature of 293K. To achieve complexes of human carbonic anhydrase II with acetazolamide, the native enzyme was soaked in a solution of dimethyl sulfoxide along with soluble acetazolamide to reach a final concentration of 0.5mM human carbonic anhydrase II and 4.5mM acetazolamide. The only ligand present was zinc (II) ion which acts as the metal cofactor for the enzyme. The structure of acetazolamide-bound human carbonic anhydrase II was determined using microcrystal electron diffraction (4).

Expasy is a hub for databases and software tools, with several relevant tools for evaluating protein properties. Using Expasy, the molecular weight of acetazolamide-bound human carbonic anhydrase II was found to be 29246 Da. The isoelectric point of the protein found to be 6.87 (5). The protein is globular and consists of a single subunit. The amino acid sequence is 257 residues long, comprising the single functional domain of the protein. The protein is fully crystallized.

The primary structure of acetazolamide-bound human carbonic anhydrase II consists of primarily polar or charged amino acid residues. There are no long stretches of hydrophobic amino acids to create a membrane-spanning alpha-helix or to cause hydrophobic collapse. The abundance of polar and charged amino acids enhance the stability and water-solubility of the protein so that it can function in an aqueous environment. The acetazolamide-bound complex doesn’t differ in amino acid sequence from the native enzyme (6). The secondary structure of the protein is composed of mostly beta sheets. 17.5% of the residues are in alpha-helices, and 23.0% of the residues are in beta sheets (6). However, there are 5 separate alpha-helix strands and 15 separate beta strands, giving the secondary structure more beta-sheet character. The remaining 59.5% of residues are interspersed between alpha-helices and beta sheets in random coils (6). Each beta strand and alpha helix contains mostly polar or charged residues. The tertiary structure of acetazolamide-bound human carbonic anhydrase II is a globular shape. The quaternary structure consists of the single subunit.

The hydration of carbon dioxide is catalyzed by the zinc-complexed carbonic anhydrase II. When complexed with any other metal or with acetazolamide, the hydration reaction does not proceed (1). Under uninhibited conditions, zinc coordinates with a hydroxyl group, acting as a nucleophilic Lewis acid, and the oxygen of the hydroxyl group attacks the carbon of carbon dioxide. (7) This substrate, which links the enzyme to carbon dioxide, then undergoes a proton transfer, with help from nearby imidazole groups acting as proton shuttles. This proton transfer creates an ester bond between the zinc and the carbon. Water then reacts with the complex and breaks the complex into bicarbonate and a water molecule coordinated with the zinc ion of the enzyme (1). This reaction scheme is depicted in Figure 1.

The active site of carbonic anhydrase II is Val-135, and His-64 is used as a proton shuttle during the activity of the enzyme in carbon dioxide hydration. Zinc metal cofactor is a ligand that coordinates with the protein at His-94, His-96, and His-119 (4). Hydrogen bonds and ionic interactions bind acetazolamide to human carbonic anhydrase II. The amino group of Gln-92 forms a hydrogen bond to the carbonyl oxygen of the acetylamido group of acetazolamide. The amide proton of acetazolamide forms a hydrogen bond to C-7 of Thr-199. The sulphonamide oxygen of acetazolamide forms a hydrogen bond to the main chain nitrogen of Thr-199. Phe-131 forms weak hydrophobic interactions with acetazolamide (2-3).

Position-specific iterated basic local alignment search tool (PSI-BLAST) is a protein comparison tool that, when given a query protein sequence, returns subject protein sequences ranked by similarity. The degree of similarity is conveyed with an E value, where any E value below 0.05 is significant. Homology between the query protein and another protein decreases the E value, gaps increase the E value, and longer sequences of homology further decrease the E value (8). While PSI-BLAST compares the primary structures of proteins, the Dali Server compares the secondary structures of proteins. Dali uses a sum of pairs method and returns a measure of similarity obtained by comparing the intramolecular distances when two structures are superimposed. Proteins with significantly similar secondary structure have Z-scores greater than 2 (9).

The comparison protein for acetazolamide-bound human carbonic anhydrase II found in Homo sapiens was carbonic anhydrase II (PDB ID: 4NCR) from Bos taurus. This protein was chosen for comparison because it carries out the same enzymatic activity in a different organism. The E value from PSI-BLAST, indicating the homology of the primary structure with acetazolamide-bound human carbonic anhydrase II, was 8 x 10-141, which is far below the threshold value of 0.05 (10). The Z-score from the Dali server, demonstrating the similarity of secondary structures, was 44.4 (11).

Both acetazolamide-bound human carbonic anhydrase II and carbonic anhydrase II from Bos taurus have an associated zinc (II) ion, a single subunit, and the same function of hydrating carbon dioxide through identical mechanisms. The amino acid sequence of the two proteins is 80.16% similar, with 206 of 257 amino acids identical between the two structures. Of the 41 amino acids that differed, 20 were conservative amino acid substitutions between the two proteins, which contributes to the functions of each protein being the same (8). Additionally, because half of the amino acid substitutions were conservative, the polarity of the structure wasn’t significantly altered, so carbonic anhydrase II from Bos taurus has the exact same secondary structure as acetazolamide-bound human carbonic anhydrase II from residue 12-257 (11).

Human carbonic anhydrase II is an essential enzyme in the human body. A deficiency in the native enzyme can result in osteoporosis, renal tubular acidosis, and brain calcification (12). The acetazolamide-bound form of human carbonic anhydrase II inhibits the enzyme activity, which leads to diseases but also has potential to treat diseases such as glaucoma. Further study could be done into the autoantibodies against carbonic anhydrase II, which have the same effect as acetazolamide binding to carbon anhydrase II. The potential of acetazolamide binding as a drug also warrants further study.