Carbonic anhydrase activator

The X-ray structure of the unsubstituted tris(pyrazolyl)borato zinc nitrate has been solved showing a unidentate coordination mode for nitrate, in contrast with the t-butyl substituted ligand, which shows anisobidentate nitrate coordination due to the steric effects.232 A partial explanation of the reduced activity of cadmium-substituted carbonic anhydrase is offered by Parkin on the basis of the comparison of nitrate coordination to cadmium and zinc trispyrazo-lylborate moieties. A contributing factor may be the bidentate coordination supported by the cadmium that does not allow the facile access to a unidentate bicarbonate intermediate, which could be highly important to carbonic anhydrase activity.233... 

Carbonic Anhydrase Activity of Human Aorta Tissue... 

Segments of human aorta obtained fresh at autopsy were freed from blood and covering connective tissue and analyzed manometrically for carbonic anhydrase activity. Twelve specimens from 7 males and 5 females yielded nearly a 9-fold spread in values (0.12 to 1.05 units).31 This is interesting in view of the zinc content of carbonic anhydrase and the extremely wide variations in the zinc content of blood plasma and spleens which have been observed (pp. 55 and 72). 

The fact that zinc is known to be a component of an enzyme carbonic anhydrase leaves no doubt as to its physiological significance. The amount of zinc in erythrocytes seems to parallel the carbonic anhydrase activity.21 The leucocytes which appear to lack carbonic anhydrase contain about 25 times as much zinc (per cell) as do the erythrocytes.20 It seems likely that an investigation of the zinc content of different types of white blood cells coupled with a study of individuals from the standpoint of the different types of white cells present (p. 35) would lead to the discovery of substantial inter-individual differences. The wide spread in the zinc concentrations in three human spleens has already been mentioned (p. 72). A recent study has been made of the intake and excretion of zinc by 13... 

Drugs of this group inhibit activity of carbonic anhydrase, an enzyme that catalyzes the reversible reaction of water and carbon dioxide, which forms carbonic acid. The mechanism of action of this group of drags is not fuUy understood. However, inhibition of carbonic anhydrase activity leads to a reduction of carbonic acid formation and an increase in bicarbonate, sodium, and potassium excretion with urine, which eventually leads to a significant increase in the process of excreting water from the organism. 

Tempering C., Scozzafava, A., Puccetti, L., and Supuran, C. (2005). Carbonic anhydrase activators X-ray crystal structure of the adduct of human isozyme II with histidine as a platform for the design of stronger activators. Bioorg. Med. Chem. Lett. 15,5136-5141. 

Subsequent to CO2 association in the hydrophobic pocket, the chemistry of turnover requires the intimate participation of zinc. The role of zinc is to promote a water molecule as a potent nucleophile, and this is a role which the zinc of carbonic anhydrase II shares with the metal ion of the zinc proteases (discussed in the next section). In fact, the zinc of carbonic anhydrase II promotes the ionization of its bound water so that the active enzyme is in the zinc-hydroxide form (Coleman, 1967 Lindskog and Coleman, 1973 Silverman and Lindskog, 1988). Studies of small-molecule complexes yield effective models of the carbonic anhydrase active site which are catalytically active in zinc-hydroxide forms (Woolley, 1975). In addition to its role in promoting a nucleophilic water molecule, the zinc of carbonic anhydrase II is a classical electrophilic catalyst that is, it stabilizes the developing negative charge of the transition state and product bicarbonate anion. This role does not require the inner-sphere interaction of zinc with the substrate C=0 in a precatalytic complex. 

The inhibition of CA II and total carbonic anhydrase activities was found below the degree of inhibition anticipated to be necessary for a pharmacological effect on renal function and respiration when dorzolamide was administered orally at 2 mg b.i.d for up to 20 weeks [14]. 

In maximal doses, chlorothiazide will markedly increase excretion of water, Na, K+, Cl and HCOj". Chlorothiazide due to its weak carbonic anhydrase activity, can cause some loss of bicarbonate in therapeutic dose. 

Other reports concerning chemically modified amino acid as metal chelating agents used for the carbonic anhydrase active site model reconstruction are in close agreement with the small contribution of Zn(II) binding to the proton chemical shift variation discussed above. NMR experiments carried out in DMSO-rfg, at 300 K, and the observed... 

Very recently the isolation of still active carbonic anhydrase from the biocrystalline layer of hens egg shells has been reported. Crude egg shell extracts exhibited no enzyme activity. however, after removal of Ca2+ and simple gel filtration of the extracts, carbonic anhydrase activity could be measured. Further purification steps led to the isolation of two isoenzymes. The possibility of inhibition of this enzyme activity by shell components cannot be excluded although recombination of shell components after separation did not inhibit that enzyme. The isolated enzymes has an apparent molecular size of 28,000 daltons. Carbonic anhydrase from egg shells catalyzes the reversible hydration of CO2 + HOH H+ + HCO3-. This probably is the primary action of the enzyme of the shell. Moreover, egg shell carbonic anhydrase catalyzes the hydration of acetaldehyde and pyridine aldehyde. Furthermore, the same enzymes have esterase activity (hydrolysis of p-nitrophenyl acetate). Whether the latter activities play a role in the egg shell cannot be judget at the present time. 

Carbonic anhydrase activities of the egg shells are inhibited by reaction with p-mercuribenzoate by blocking reactive SH-groups of the enzyme. Omission of 2-mercaptoethanol from buffer solutions for extraction and fractionation also resul-... 

The method has been applied widely using, for example, methylamine. A newer method employs an isotope exchange procedure to measure the pH-sensitive carbonic anhydrase activity naturally present in mitochondria.181... 

There is general agreement that carbonic anhydrase activity is linked to the zinc ion and its ligands. At the active site water is bound to the zinc but the state and involvement of the water in the actual catalysis is a matter of controversy. According to a widely accepted opinion the zinc-bound water is ionized and the activity is a function of pH1S6. ... 

Deposition of mineral matter is limited by diffusion of calcium and/or phosphorus to the site of deposition400-. Since the transfer of both compounds is enzymatically controlled (see p. 21) equilibrium relationships may change environmental settings critical for the deposition of minerals. For instance, by limiting carbonic anhydrase activity in one direction, a pool of H2C03 may build up at the site of deposition in the reverse situation HC03 will concentrate. As a consequence,... 

Figure 7.11 Schematic of the carbonic anhydrase active site showing the Zn(ll) cation coordinated to a benzenesulfonamide (ArS02NH2) inhibitor molecule.

Inhibition of carbonic anhydrase activity profoundly depresses bicarbonate reabsorption in the proximal tubule. At its maximal safely administered dosage, 85% of the bicarbonate reabsorptive capacity of the superficial proximal tubule is inhibited. Some bicarbonate can still be absorbed at other nephron sites by carbonic anhydrase-independent mechanisms, and the overall effect of maximal acetazolamide dosage is about 45% inhibition of whole kidney bicarbonate reabsorption. Nevertheless, carbonic anhydrase inhibition causes significant bicarbonate losses and hyperchloremic metabolic acidosis. Because of this and the fact that HCO3" depletion leads to enhanced NaCl reabsorption by the remainder of the nephron, the diuretic efficacy of acetazolamide decreases significantly with use over several days. 

Landolfi et al. (1997) reported a modified procedure for the measurement of carbonic anhydrase activity. The measure of carbonic anhydrase activity is based on the rate of CO2 hydration by the enzyme. Such transformation was monitored by a procedure which consists of the measure of time necessary for the pH of an appropriate buffer to decrease from 8 to 7.5 in the presence of a constant CO2 flow this time period is dose-dependently reduced by the addition of the enzyme and further modified in the presence of carbonic anhydrase inhibitory compounds. 

Landolfi C, Marchetti M, Ciocci G, Milanese C (1997) Development and pharmacological characterization of a modified procedure for the measurement of carbonic anhydrase activity. J Pharmacol Toxicol Meth 38 169-172 Maren TH (1960) A simplified micromethod for the determination of carbonic anhydrase and its inhibitors. J Pharmacol Exp Ther 130 26-29... 

Carbonic anhydrase inhibitors, such as acetazolamide and dichlor-phenamide, act as diuretics to increase excretion of water by inhibiting carbonic anhydrase activity. This in turn leads to a reduction in the level of bicarbonate in aqueous humour. 

Supuran CT, Barboiu M, Luca C, Pop E, Dinculescu A (1996) Carbonic anhydrase activators. Part 14. Syntheses of positively charged derivatives of 2-amino-5-(2-aminoethyl) and 2-amino-5-(2-aminopropyl)-l,3,4 thiadiazole and their interaction with isozyme II. Eur J Med Chem 31 597-606... 

Davis, Robert R, The Measurement of Carbonic Anhydrase Activity. 11 307... 

Inhibition of carbonic anhydrase activity in the ciliary processes is probably the mechanism responsible for decreased aqueous formation produced by CAIs. The production of bicarbonate in the ciliary epithelium appears to play a key role in the formation of aqueous humor. Bicarbonate is the key anion associated with the decrease in aqueous formation produced by inhibition of carbonic anhydrase. 

Figure 9.23. Effect of pH on Carbonic Anhydrase Activity. Changes in pH alter the rate of carbon dioxide hydration catalyzed by carbonic anhydrase II. The enzyme is maximally active at high pH.

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