Carbonic anhydrase anion binding

XH NMR data of copper-carbonic anhydrase (CuCA) complexes in the presence of different anions indicated that water is present in the coordination sphere along with the anions (137). The three histidines, the anion, and the coordinated water molecule arrange themselves to maintain essentially a SQPY. His-94 would be in the apical position of the SQPY and two other histidine residues (His-96 and His-119) along with the anion and the coordinated water are positioned in the basal plane. Most likely the anion is present in the hydrophobic pocket or in the site and the coordinated water molecule is present in the C site or the hydrophilic binding site. 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

Carbonic anhydrase is inhibited by monovalent anions. For the bovine enzyme, apparent binding constants range from about 1 M-1 for F to 5 x 105 M 1 for HS at 25° C and pH 7.5 (Table 4). The interaction... 

The presence of imidazole groups in the active site region of human carbonic anhydrase B has, in fact, been demonstrated by chemical modification. Thus, bromoacetate reacts specifically with the 3 -N of a histidine residue to give a partially active monocarboxymethyl enzyme (65). The reaction depends on the initial combination of the bromoacetate ion with the anion binding site (65,83). In a detailed study, Bradbury (83) has shown that the irreversible reaction at saturation with iodoacetate... 

The idea of H2C03 as substrate for carbonic anhydrase is strongly supported by inhibitor binding studies158, 159. Small anion or sulfonamide inhibitors are linked to the zinc ion at high pH. The complex picks up a proton and the inhibitor is bound in a neutral form. 

Coleman, J. E. Mechanism of action of carbonic anhydrase, substrate, sulfonamide, and anion binding. J. Biol. Chem. 242, 5212—5219 (1967). 

Taylor, P. W., and Burgen, A. S. V. Kinetics of carbonic anhydrase inhibitor complex formation. A comparison of anion- and sulfonamide-binding mechanisms. Biochemistry JO, 3859-3866(1971). 

The ability of zinc in carbonic anhydrase to become five-coordinate is also confirmed by the structural studies on enzyme-inhibitor complexes discussed in Section 62.1.4.2.1. There is much evidence for the coordination of anionic inhibitors to the metal, while the competitive inhibitor imidazole gives a five-coordinate centre. Sulfonamides are powerful inhibitors which bind directly to the zinc and also interact with the protein. The sulfonamide acetazolamide has significant affinity for the apoenzyme. It is probable " that the first interaction between the enzyme and aromatic sulfonamides is a hydrophobic interaction between the aromatic ring and residues in the active site cavity, followed by ionization of the SO2NH2 group prior to complex formation. Sulfonamides only bind to the zinc and cobalt enzymes, i.e. the two metals that give an active enzyme. 

Anions are attracted in the metal cavity by the positive Zn(N30H2) moiety, and are believed to bind to zinc in carbonic anhydrase very effectively so their use should be avoided as much as possible if the goal is to study the enzyme as it is. When the protein is dialyzed against freshly doubly distilled or carefully deionized water under an inert atmosphere, the pH of the sample approaches the isoelectric point, which is below 6 for HCA I and bovine (BCA II) enzymes. The pH can then be adjusted by appropriate additions of NaOH. All the measurements reported in the literature performed in acetate, phosphate, imidazole, or tris sulfate buffers are affected by the interference of the anion with the metal ion. However, buffer species containing large anions like Hepes (4[(2-hydrox-yethyl)-l-piperazinyl]ethanesulfonic acid) can be used," since these anions do not enter the cavity. 

Once inside the tubular cell, there is a possibility of cytoplasmic binding or distribution into intracellular compartments. For example, the extensive renal accumulation of methazolamide in the kidney model was attributed to drug complexation with cytoplasmic carbonic anhydrase. Furthermore, biotransformation (most notably phase II metabolism) within the kidney cell is also possible. The role of intracellular distribution and metabolism on the renal disposition of organic anions requires further study. 

It is well documented in the literature that the sulfonamide anion (SO2NH") specifically binds to the carbonic anhydrase and inhibits its activity. This sulfonamide anion forms a complex with zinc cation at the active... 

In the renal buffering process, sodium (Na+) is exchanged for hydrogen ions (H+) and binds with some of the bicarbonate (NaHCOj), which later breaks down again as Na" is actively removed through a Na - K+ mechanism (discussed in more detail in Chapter 5). The H" ions are bound with carbonic anhydrase on the border of the proximal tubules of the kidneys, which convert the H first to H COj and then to H O and CO. Some H+ ions also bind with the ammonia (NHj) produced in the kidneys as a result of amino acid catabolism and an abundant anion found in the glomerular filtrate, chloride (CT), to form ammonium chloride (NH Cl), a weak acid that is excreted in the urine. Thus it is clear that other electrolytes are involved in the acid-base balancing process and can be affected by acid-base imbalances. These impacts will be discussed with each electrolyte. 6... 

Ward and Cull [429] investigated the binding of a number of anions to bovine carbonic anhydrase by observing their effect on the 35... 

Nome et al. [4Z1] recently reported on the competition between Cl" and the non-coordinative anion Au(CN)2 for binding to human carbonic anhydrase B and C and this observation suggests that the halide ion binding may be of a non-coordinative type. 

The schemes presented above offer a consistent rationalization of the binding of substrate and inhibitors to carbonic anhydrase under a broad range of conditions, and are generally consistent with earlier, more limited studies of the anionic inhibition of the Zinc(II) carbonic anhydrase catalyzed hydration of CO2 and dehydration of HC03(3,20). Carbonic anhydrase catalyzed esterase activity do s not conform to this scheme, unless the binding represented by Kj is eliminated or greatly reduced. Esterase activity inhibition studies have shown that even the most potent anionic inhi-... 

We propose that the catalysis of Zinc(II) carbonic anhydrase proceeds through a variety of four- and five-coordinate Zinc(II) species in rapid equilibrium. The extensive and stimulating series of spectral studies recently undertaken by Bertini and colleagues (30-32) has strongly suggested to us that the expansion of the coordination sphere of metal-substituted carbonic anhydrase is an important mode of accommodation of anions in the enzyme active site. X-ray evidence(23) and Cl NMR data(34) as well as other spectral studies, have localized the binding of simple anionic inhibitors to the inner coordination sphere of the zinc atom in the acidic form of carbonic anhydrase. 

It has been noted previously(22) that HCN and other ligands supplying an exchangeable proton bind to carbonic anhydrase by donation of a proton and association as an anion. We attribute the ability of HCO3 to bind to the basic form of carbonic anhydrase to this effect. The relevant equilibrium is... 

An important consideration in any proposed scheme is the rationalization of the uncompetitive inhibition of carbonic anhydrase at high pH. We suggest that CO2 itself acts as an electron sink for Zn-OH, and thus its presence facilitates anion binding, acting in a Lewis acid sense much as the HCO3 proton participates as a Br nsted acid ... 

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