Carbonic anhydrase anionic inhibition

Carbonic anhydrase is inhibited by several classes of inhibitor. The sulfonamides, of general formula XS02NH2, are well known, while inhibition by simple anions occurs particularly at higher pH values. Imidazole is a unique example as it is the only compound known at present to act as a competitive inhibitor for the hydration of C02. A great deal of mechanistic information has come from a study of the action of these compounds. 

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... 

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... 

Carbonic anhydrase plays an important role in the secretion of aqueous humor [1,2]. This enzyme was first demonstrated to be present in the ciliary processes of the rabbit, and its presence was later confirmed in human ciliary processes [3,4]. Carbonic anhydrase is responsible for the generation of bicarbonate anions which are secreted from the ciliary process into the posterior chamber, with sodium being the counter ion. Inhibition of carbonic anhydrase in the ciliaiy processes of the eye decreases aqueous humor secretion, presumably by slowing the formation of bicarbonate ions with subsequent reduction in sodium and fluid transport. The role of the enzyme in aqueous humor secretion has been reviewed in detail by Maren [1]. 

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. 

After I had studied the secretion of bromide and iodide, it was obvious that the next anion I should study was thiocyanate. I fed my pouch dogs sodium thiocyanate, stimulated them to secrete by subcutaneous injections of histamine, and attempted to collect their gastric juice. I was frustrated by the scantiness of secretion, and eventually I realized that thiocyanate was inhibiting acid secretion. My friend Joseph F. Ross, who was also a fellow in pathology at Rochester, suggested that thiocyanate might be inhibiting carbonic anhydrase. I made the appropriate test and found that the enzyme is indeed inhibited by thiocyanate. 

METALLOENZYMES AND MODEL SYSTEMS. CARBONIC ANHYDRASE SOLVENT AND BUFFER PARTICIPATION, ISOTOPE EFFECTS, ACTIVATION PARAMETERS AND ANIONIC INHIBITION... 

The inhibition of carbonic anhydrase by anions has long been recognized. The inhibitory effect of Cl was noted by early investi-gators(19). A later study of anion inhibition by stopped-flow techniques was compromised by the presence of 80mM Cl in buffers used (20). The inhibition of the hydrase activity of the Cobalt(II) substituted enzyme has been investigated over the full pH range(21). Anionic inhibition of esterase activity has been studied by initial rate techniques(11-13) and by complexometric titration(22). None of the work thus far published has included full scale Michaelis-Menten analysis of the inhibition of the native Zinc(II) enzyme towards its natural substrate over an extended pH range. 

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-... 

A study of the anion inhibition of Cobalt(II) HCAC CO2 hydration activity reported a loss of inhibitory power of anions at high pH(21). Further scrutiny of the high pH anion inhibition of metal substituted carbonic anhydrase is certainly in order. If, as it appears, high pH anion activity inhibition is a property confined to the natural substrate of the Zinc(II) enzyme, the implications for conclusions drawn from studies of model substrates and metal substituted enzymes need to be carefully scrutinized. 

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 ... 

Components of the gastric barrier. Illustrated is a mucous coat, which acts as an unstirred layer, inhibiting fast diffusion of secreted HCOs" via an apical anion exchanger. The basal-lateral surface has pH regulatory mechanisms, such as sodium proton exchange or sodium-dependent entry of bicarbonate. The necessary Na gradient is driven by the sodium pump, and bicarbonate is formed by the action of carbonic anhydrase (CA). 

Characterization and anions inhibition studies of an a-carbonic anhydrase from the teleost fish Dicentrarchus labrax. Bioorganic Medicinal Chemistry, 19, 744-748. 

Biochemical Aspects. The zinc-containing metalloenzyme carbonic anhydrase (carbonic hydro-lyase, EC 4.2.1,1) is specifically inhibited by univalent anions and by aromatic sulphonamides. AfT (25 for the binding reactions between bovine carbonic anhydrase and 3-methyl-2-acetylimino-l,3,4-thiadiazoline-5-sulphonamide (Methazolamide) is — 59.0kJmol and that for 5-phenyl-sulphonamido-l,3,4-thiadiazole-2-sulphonamide (CL 11366) is — 57.7kJmol" The thermodynamic quantities did not correlate with the assumed structural features of the binding process. ... 

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