Carbonic anhydrase and zinc

The zinc acetate complex of tris(3-/-butyl-5-methylpyrazol-l-yl)borate was prepared as a structural model for carbonic anhydrase and comparison with the enzyme active site structures confirmed that the complexes are excellent structural models.239 A mononuclear zinc hydroxide complex can also be formed with the tris(pyrazolyl) borate ligand system as a structural model for carbonic anhydrase.240... 

The ligand tris[2-(l-methylbenzimidazol-2-yl)ethyl] nitromethane (25) has been used in the formation of zinc complexes as models of the active site of carbonic anhydrase, and the formed complexes reveal affinity for the sulfonamide-containing enzyme inhibitor acetazolamide.248... 

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). 

Zinc is a microelement essential for proper functioning of the human body. The level of daily demand for zinc was established as 13 to 16 mg (Ziemlahski, 2001). Zinc plays a role in protein and carbohydrate metabolism and is a component of over 60 metaloenzymes, including alkaline phosphatase, pancreatic carboxypeptidases A and B, alcoholic and lactic dehydrogenases, carbonate anhydrase, and proteases. It also forms bonds with nucleic acids -which is very important for their functioning (Prasad, 1983 Valee and Falchuk, 1993). 

Figure 7.1 (a) The denatured conformation of the zinc metalloenzyme carbonic anhydrase and the ESI mass spectra obtained under acidic denaturing conditions, (b) The ESI mass spectra obtained under native-state conditions. The decon-voluted ESI mass spectra of carbonic anhydrase reveals the protein molecular weight. The three dimensional structure is protein Data Bank ID IBNl. 

The results of site-directed mutagenesis analysis of zinc ligands of higher plant p-carbonic anhydrase and of P. purpureum carbonic anhydrase have confirmed that zinc is essential for catalysis. X-ray fine structure data indicated that a water molecule is hydrogen bonded to the zinc-ligated Asp-151 and Asp-405. The water molecule is not directly coordinated to the zinc atom. A possible catalytic mechanism of C02 hydration cycle (211) has been proposed as given in Scheme 10. 

Eiichi Kimura is retired from the Department of Medicinal Chemistry at Hiroshima University in Japan. His recent research interests have included the supramolecular chemistry of macrocyclic polyamines and their use in molecular recognition and as zinc-enzyme models. These interests have led to the development of fluorophore sensors for Zn(II) [8] use of macrocycles to effect selective recognition of anions [9], nucleobases in polynucleotides [10], thymidine mono- and diphosphate nucleotides (11), carbonic anhydrase and carboxypeptidase [12], and development of Zn(II)-macrocycle anti-HIV agents [13], In May 2004, he received a Purple Ribbon Award from the Emperor of Japan. 

It appears that cobalt plays a particularly important role in the growth of cyanobacteria (Saito et al, 2002 Sunda and Huntsman, 1995b). Both Prochlorococcus and Synechococcus show an absolute cobalt requirement that zinc cannot substitute for (Figure 18(a)). The growth rate of Synechococcus is little affected by low zinc concentrations, except in the presence of cadmium which then becomes extremely toxic (Saito et al, personal communication). The biochemical processes responsible for the major cellular utilization of zinc and cobalt in marine cyanobacteria are unknown, however. These metals may be involved in carbonic anhydrase and/or other hydrolytic enzymes. Cobalamin (vitamin B12) synthesis is a function of cobalt in these organisms, yet B12 quotas tend to be very small (on the order of only 0.01 p.mol (mol C) ) and hence are not likely represent a significant portion of the cellular cobalt (Wilhelm and Trick, 1995). 

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. 

One of the most important discoveries concerning the biological role of zinc occurred in 1940 when Keilin and Mann showed that zinc is an essential compound of erythrocyte carbonic anhydrase, an enzyme cata-lytically involved in the transport of CO2 in blood (6). Following the 70-year interval between the initial recognition of a metabolic zinc deficiency and the characterization of the first zinc metalloenzyme, there was a period of about 15 years before the second zinc enzyme was identified. In 1955, Vallee and Neurath reported that carboxypeptidase A from bovine pancreas contained 1 g-atom Zn per mol of protein and was essential to the function of the enzyme (7). The presence of zinc in carbonic anhydrase and carboxypeptidase A indicated that a primary role of zinc would be to function in zinc metalloenzymes (62). However, it seemed unhkely that disrupting the activity of carboxypeptidase A or carbonic anhydrase would have profound eflFects on growth. 

Another catalytic principle can be learned from carbonic anhydrase and two other zinc enz3rmes which have been well-characterized carboxypeptidase A and liver alcohol dehydrogenase. 

Zinc is recognized as essential to all forms of life, and is the most common transition metal in the body after iron. There are 2 to 3 g of zinc in adults, compared with 4 to 6 g of iron and 0.25 g of copper. Enzymes containing zinc include carbonic anhydrase and carboxypeptidase, the first two metalloenzymes detected - now there are over 300 zinc enzymes known. Zinc serves an important structural role in DNA binding proteins, stabilizing the correct binding site. Zinc reserves are stored in the metallothionine proteins. 

Comparison of the zinc(II) ion environment of the enzyme carbonic anhydrase and of a small molecule model that employs a triaza-macrocycle to supply the N-donor ligand set. 

Enzymes having metals as their components can also be inhibited by a substitution of one of these metal ions by another ion with the same charge and a similar size. For example, the toxic effect of cadmium is due to a substitution for zinc, which is a common component in metalloenzymes. The Zn " " and Cd ions are chemically similar, however, the cadmium-containing enzyme does not function properly. The Cd " ions can result, for instance, in the inhibition of amylase, adenosine triphosphatase, adcohol dehydrogenase, glutamic-oxalacetic transaminase, carbonic anhydrase and peptidase activity in carboxypeptidase [4]. 

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