Carbonic anhydrase apoenzyme

The zinc can be removed from carbonic anhydrase by dialysis against certain chelating agents, notably pyridine-2,6-dicarboxylic acid.499 The apoenzyme can be reconstituted by a number of... 

Zinc can be removed from carbonic anhydrase on dialysis against a chelating agent at pH about 5 (37, 38). The apoenzyme is inactive but the gross conformation of the protein is maintained (37, 39, 40). The metal-chelating site can accomodate any of the divalent transitional metal ions from Mn2+ to Zn2+ as well as Cd2+ and Hg2+ (38,41). Most of these metallocarbonic anhydrases have low activities or are inactive, however. Only Zn2+ and Co2+ are efficient activators. As shown in Table 3, this narrow metal-ion specificity is observed for the CO 2 hydration as well as for the esterase reactions. 

Use of apoenzymes for the detection of metal ions Generally, apoenzymes of metalloenzymes can be used for the detection of the corresponding metal ion. Restoration of enzyme activity obtained in the presence of the metal ion can be correlated to its concentration. This principle has been demonstrated in the detection of copper while evaluating reconstituted catalytic activities in galactose oxidase and ascorbate oxidase and also in the detection of zinc since this ion is essential for the activity of carbonic anhydrase and alkaline phosphatase [416]. The need of stripping the metal for the preparation of the apoenz5une may demand tedious procedures and a catalytic assay with the addition of the substrate is always required for detection. 

Zinc is involved in many biochemical functions. In 1940 Keilin and Mann (11) reported for the first time that carbonic anhydrase was a zinc metalloenzyme. Over the next 20 years, only five additional zinc metallo-enzymes were identified, but in the past 15 years, the total number of related enzymes from different species is more than 70 (12). Besides its role in the functions of enzymes, recently it has been shown that zinc may be involved in maintenance of structures of polynucleotides, apoenzymes, and biomembranes (13,14). 

Reduced activity of carbonic anhydrase, another zinc metalloenzyme, has been reported in gastric and intestinal tissues and in erythrocytes when the activity of the enzyme was expressed per unit of erythrocytes (91), Recently in sickle-cell-disease patients, an example of a conditioned zinc-deficient state, the content of carbonic anhydrase in the red cells was found to be decreased, correlating with the zinc content of the red cells (10,75). Inasmuch as the technique measured the apoenzyme content, it appears that zinc may have a specific eflFect on the synthesis of this protein by some mechanism yet to be understood. 

Numerous metalloenzymes have the ability to remain functional even after the metal, which presumably is present at their active center, has been replaced by another metal (13). Thus in zinc deficiency, if the apoenzyme is synthesized, as has been observed in the case of . coli alkaline phosphatase (13), then other metals which might have accumulated or are normally within the cell could substitute for zinc and generate an active enzyme. Although this is a possibility in the case of microorganisms, it certainly does not appear to be true in the case of experimental animals and man, in that the apoenzymes of alkaline phosphatase, carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase, and de-oxythymidine kinase do not accumulate in zinc-deficient tissues. Thus, one may conclude that a deficiency of zinc does specifically aflFect the activities of zinc-dependent enzymes in sensitive tissues. 

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. 

Many enzymes are conjugated proteins, formed by combination of a simple protein, called the apoenzyme (apo means off, separated from), and one or more other molecules or ions, called coenzymes. Some coenzymes are metal ions. An example is the carbonic anhydrase in human erythrocytes. The apoenzyme has molecular mass 28,000 d. It combines with one zinc ion, Zn" " , to form the active enzyme, which catalyzes the decomposition of carbonic acid to water and carbon dioxide. Another example is the amylase in human saliva, which helps digest starch. It has molecular mass 50,000 d, and requires one calcium ion, Ca" " , as coenzyme. Many enzymes have vitamins or derivatives of vitamins as coenzymes. 

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