Anhydrase CA

Carbonic Anhydrase (CA).—There has been continued interest in the mode of action of this widely found zinc-containing enzyme which catalyses the reversible interconversion of CO2 and HCOa . In a buffered solution not far from neutrality, the stoicheiometry of the reaction is 

The catalysis by CA of the exchange of between CO 2 and water at chemical equilibrium has been measured. To explain the observation that, near pH 6 and in the presence of excess CO2, the rate of catalysed hydration is less than the known initial velocity of hydration in non-equilibrium experiments, the authors suggest that removed from bicarbonate during a catalysed dehydration step at low pH has a long lifetime in the active site compared with the turnover time for one catalytic cycle. As a result of these and other studies at higher pH, Tu and Silverman suggest the following scheme  

The role of Zn in the folding of bovine CA-B has been investigated by various physicochemical techniques. The metal appears to stabilize part of the molecule and enable the whole protein to act as a more co-operative unit but the authors feel that its presence is not vital for the enzyme to fold in the correct manner. 

The rate and equilibrium constants have been reported for the binding of a series of sulphonamides to human CA-C. In all cases the increase in binding constant in a homologous series is due mainly to an increase in the association rate constant, as shown in Table 1 for 4-alkylsulphonamides. (Similar trends were found with other 

4-substituted benzenesulphonamides, with 3- and 2-substituted benzenesulphon-amides, and with thiophen sulphonamides in the case of the meta- and ortho-substituted compounds, the markedly lower binding constants were also due mainly to lower association rate constants.) It is argued that the sulphonamides bind initially to the enzyme without co-ordinating to the metal and that the initial complex formed then isomerizes into one in which complexation with the zinc has occurred. On the other hand, in the binding of the chromophoric sulphonamide (7a) to bovine CA, the rapid kinetics of the induced c.d. and difference spectra proceed in parallel (with a 

Linden, D. Chappelet-Tordo, and M. Lazdunski, Biochim, Biophys. Acta, 1977, 483, 100. Y. Pocker and D. W. Bjorkquist, Biochemistry, 1977, 16, 5698. 

Tu and Silverman have extended their studies of the effect of buffers on the CA-catalysed exchange of 0 between CO 2 and water to a comparison between three isoenzymes of CA - namely, bovine CA, human CA-B, and human CA-C. The results are consistent with the hypothesis that buffer-facilitated proton transfer enhances the catalysis in all three cases. 

The binding of the sulphonamide Neoprontosil (15) to CA has been studied by the resonance Raman and stopped-flow methods and it has been shown that (15) binds as —SO2NH2 rather than as —SOaNH . The pH dependence of the kinetics of binding is similar to the established pattern for sulphonamide inhibition of CA esterase activity and it is suggested that an outer-sphere complex is formed with the 

As in previous review periods, there have been several studies on CAs in which the zinc in the native enzyme has been replaced by a bivalent metal ion with a more convenient electronic configuration. Harrington and Wilkins have reported the stability and rate constants for the interaction of acetazolamide (16) and the 4-nitrothiophenolate ion (NTP) with the Mn-, Co-, Ni-, Cu-, and Cd-forms of bovine CA (Table 11). The differing stabilities of the various (NTP) and, especially, (16) 

Merrill, and Henkins have used C n.m.r. spectroscopy to study the binding of CO2 and HCOs to Co -human CA-B. They find that the distance between the metal ion and the atom in the complex is essentially constant (ca. 3.5 A) in the pH range over which the substrate changes from 97 % HCOa to 87 % COg. This suggests either that the relative amounts of bound COg and HCOg are pH independent (the actual amounts are not known), so that the same weighted average distance is obtained at all pH values, or that the distances of bound CO2 

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Carbonic anhydrase (CA) exists in three known soluble forms in humans. All three isozymes (CA I, CA II, and CA III) are monomeric, zinc metalloenzymes with a molecular weight of approximately 29,000. The enzymes catalyze the reaction for the reversible hydration of C02. The CA I deficiency is known to cause renal tubular acidosis and nerve deafness. Deficiency of CA II produces osteopetrosis, renal tubular acidosis, and cerebral calcification. More than 40 CA II-defi-cient patients with a wide variety of ethnic origins have been reported. Both syndromes are autosomal recessive disorders. Enzymatic confirmation can be made by quantitating the CA I and CA II levels in red blood cells. Normally, CA I and CAII each contribute about 50% of the total activity, and the CAI activity is completely abolished by the addition of sodium iodide in the assay system (S22). The cDNA and genomic DNA for human CA I and II have been isolated and sequenced (B34, M33, V9). Structural gene mutations, such as missense mutation, nonsense... 

The carbon dioxide produced during cellular metabolism diffuses out of the cells and into the plasma. It then continues to diffuse down its concentration gradient into the red blood cells. Within these cells, the enzyme carbonic anhydrase (CA) facilitates combination of carbon dioxide and water to form carbonic acid (H2C03). The carbonic acid then dissociates into hydrogen ion (H+) and bicarbonate ion (HC03). 

Figure 2. Sodium and chloride uptake across an idealised freshwater-adapted gill epithelium (chloride cell), which has the typical characteristics of ion-transporting epithelia in eukaryotes. In the example, the abundance of fixed negative charges (muco-proteins) in the unstirred layer may generate a Donnan potential (mucus positive with respect to the water) which is a major part of the net transepithelial potential (serosal positive with respect to water). Mucus also contains carbonic anhydrase (CA) which facilitates dissipation of the [H+] and [HCO(] to CO2, thus maintaining the concentration gradients for these counter ions which partly contribute to Na+ import (secondary transport), whilst the main driving force is derived from the electrogenic sodium pump (see the text for details). Large arrow indicates water flow...

Carbon-graphite fibers, 24 614 Carbon-graphite materials, 12 747-748 Carbon-half moly, 17 16 Carbonic acid, 4 805 6 290 Carbonic anhydrase (CA) inhibitors, antihypertensives, 5 169 Carbonic anhydrase volume profile,... 

Carbonic anhydrase (CA, also called carbonate dehydratase) is an enzyme found in most human tissues. As well as its renal role in regulating pH homeostasis (described below) CA is required in other tissues to generate bicarbonate needed as a co-substrate for carboxylase enzymes, for example pyruvate carboxylase and acetyl-CoA carboxylase, and some synthase enzymes such as carbamoyl phosphate synthases I and II. At least 12 isoenzymes of CA (CA I—XII) have been identified with molecular masses varying between 29 000 and 58 000 some isoenzymes are found free in the cytosol, others are membrane-bound and two are mitochondrial. 

The first explicit treatment of the DCC concept using proteins was reported by Lehn and Hue in 1997 [1,7]. In a touchstone paper for the development of the DCC area in general, they synthesized an imine DCL to probe the active site of the enzyme carbonic anhydrase (CA). The composition of... 

Figure 7.2 The mass-to-charge difference (Am/z) between the peaks for the nnbound carbonic anhydrase (CA) protein and the CA-inhibitor complex can be mnltiplied by the charge state to give directly the molecnlar weight of the CA-...

Concomitant oral carbonic anhydrase inhibitors There is a potential for an additive effect of the known systemic effects of carbonic anhydrase (CA) inhibition in patients receiving an oral and ophthalmic CA inhibitor. The concomitant administration of ocular and oral CA inhibitors is not recommended. 

Carbonic anhydrase-medlated Na+/H+ exchange In proximal convoluted tubule. Na+/H+exchange across apical cell membranes Is shown by the open circle. Carbonic anhydrase (CA) is present in a membrane-bound form in the apical membrane and a soluble form within the cytoplasm. The Na+/K+-ATPase is shown by the filled circle at the basolateral membrane. 

In vitro studies indicated that dorzolamide binds strongly to erythrocyte carbonic anhydrase CA II and very weakly to CA I. The desethyl metabolite also binds to both CA I and CA II, but has less affinity for CA II and is considerably less selective for CA II than dorzolamide. 

Figure 12.2 Mechanism of action of carbonic anhydrase inhibitors on the proximai convoiuted tubuie. Carbonic anhydrase is an enzyme that cataiyses the interconversion of C02and H20 to H2C03and is found in the iuminai epitheiium of the proximai, and to a iesser extent, the distai convoiuted tubuie. It is essentiai for the conservation of body base in the form of HCO-3. An antiporter (1) mechanism (the movement of substances across a barrier in opposite directions) exchanges fiitrate Na+for ceiiuiar H+. The H+combines with fiitrate HCO-3to form carbonic acid which is converted to C02and H20 in the presence of carbonic anhydrase (CA). The C02is reabsorbed by the ceii thereby conserving HCO-3. Acetazoiamide inhibits the activity of carbonic anhydrase and limits the conversion of HCO-3to absorbable C02. The concentration of HCO-3in the filtrate increases as does the urinary loss. P, the sodium pump ECF, extracellular fluid.

Carbonic anhydrase (CA, EC 4.2.1.1), the first enzyme recognized to contain zinc , is ubiquitous. It occurs in animals, plants and bacteria. The essential physiological function... 

Apical membrane Na+/H+ exchange (via NHE3) and bicarbonate reabsorption in the proximal convoluted tubule cell. Na+/K+ ATPase is present in the basolateral membrane to maintain intracellular sodium and potassium levels within the normal range. Because of rapid equilibration, concentrations of the solutes are approximately equal in the interstitial fluid and the blood. Carbonic anhydrase (CA) is found in other locations in addition to the brush border of the luminal membrane. 

Carbonic anhydrase (CA), in particular, has proved a popular target for the development of structural models. The role of this enzyme is to catalyse the simple, but very important, reaction of C02 fixation (Equation 12.1). 

By the use of a model system, Kimura et al. [17] tried to mimic the function of the two mechanistically most typical zinc(II) enzymes. Carbonic anhydrase (CA, EC 4.2.1.1) catalyses the reversible hydration of carbon dioxide to bicarbonate ion and its zinc(II) active site is bound to three histidine residues and a water molecule. Carboxypeptidase A (CPA, EC 3.4.17.1) catalyses the hydrolysis of the hydrophobic C-terminal amino acids from polypeptides, and its active-site zinc(II) is bound to two histidine residues, a glutamine residue and a water molecule which is hydrogen bound to a glutamine residue (Scheme 19). 

The reaction mechanism of carbonic anhydrase (CA) is still a matter of controversy. Pocker and Guilbert232 have reported that the hydrolysis of diesters by CA, specifically the hydrolysis of methyl 4-nitrophenylcarbonate, is similar to that which occurs in CO 2 hydrolysis the rate varies as though dependent on the ionization of a group in the enzyme with a pK near 7. Only the basic form is active. - ... 

Carbonic anhydrase (CA EC 4.2.1.1) is a zinc-metalloprotein. It catalyses the reversible hydration of carbon dioxide to bicarbonate and hydrogen ions and might be important in providing C02 for photosynthesis, although its role is somewhat controversial (Sandmann and Boger, 1983). 

Figure 7.9 Delivery of oxygen to peripheral tissue by oxyhemoglobin. Production of protons from COz and HzO, catalyzed by carbonic anhydrase (CA) and their uptake by hemoglobin are also shown. The pC02 in peripheral tissues is high and p02 is low, causing the conversion of hemoglobin R forms into the T forms with the concomitant release of 02. (Reproduced by permission from Bunn HF, Forget BG. Hemoglobin Molecular, Genetic, and Clinical Aspects. Philadelphia WB Saunders, 1986, p. 41.)...

It is this local character of vibrational dephasing that is utilized in the experiments described in this section. In these experiments, spectral diffusion of test molecules bound to enzymes has been investigated in order to study the fluctuations of the reactive sites. The local character of these interactions can be pictured in great detail since in many cases high-resolution x-ray structure of the complexes are available. One example we have studied, shown in Fig. 8, is azide bound to carbonic anhydrase (CA-N3 ) (107). Carbonic anhydrase is a zinc enzyme that catalyzes the interconversion of... 

Figure 8 The structure of the binding pocket of azide bound to carbonic anhydrase (CA-N3 ) (107). The atoms of the azide ion, which is bound to the active site (Zn+2), is in close contact with Thr-199 (indicated by the dotted lines). (From...

Albizziine (ureido amino acid) Carbonic anhydrase (CA) Phenolic... 

Carbonic anhydrases (CA) have been one of the early addressed biological targets for which the DCC [49-53] may offer a complementary route to high-throughput combinatorial methods [54],... 

Figure 3 Zinc ligands of mammalian carbonic anhydrases, CA I-V. Names denote type and species of CA. Data obtained using the sequence of human CA I and the resources of the EMBL computer center ...

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