Anhydrase

Carbonic Anhydrase.—Strandberg and his colleages have calculated a 2 A resolution electron-density map of human erythrocyte carbonic anhydrase C. A polypeptide chain of 258 amino-acid residues is indicated, and two sequenced fragments have been fitted in positions 1—88 and 224— 258. The structure has a gross shape of 41 x 41 x 47 A and is built essentially of three layers. Seven distorted sections of right-handed a-helix are in the surface layers. In contrast the middle layer is an extensive mainly antiparallel pleated sheet structure with a total twist of 220° and comprising 37% of the total residues. There are aromatic regions between the central sheet and the surface layers. 

The active site is a 15 A deep conical cavity between the pleated sheet and the upper surface layer. The zinc atom is bound to of His-93 and His-95, and of His-117 (part of the pleated sheet). The distorted tetrahedral co-ordination is completed by a water molecule which is also bound to the of Thr-197. The active site region also implicates two histidines, His-63 and His-128, which lie on opposite sides of the entrance to the cavity. There 

A series of difference Fourier maps has been used to study the replacement of zinc with other metal ions and the nature of interaction of sul-phonamides and certain anions with carbonic anhydrase C. 

The two enzyme systems most frequently mentioned in connection with biomineralization are (1) carbonic anhydrase and (2) alkaline phosphatase. Little information, however, has been presented on their specific role in the deposition of minerals. Recent advances in the field of biochemistry may shed new light on this intriguing problem. 

The enzyme is quite versatile in that it catalyses the interconversion of C02 and HCO3 in solution, the hydration of aliphatic aldehydes149, pyridinecarboxylalde-hydes and pyruvic acid150, and acts as esterases with respect to certain monoesters of carboxylic acids and diesters of carbonic acid151,1S2. Carbonic anhydrase has been found in a number of animal tissues, plants and a few bacteria141.  

The catalytic mechanism for C02 hydration-dehydration by carbonic anhydrase represents the focal issue of the present discussion. We have to consider two aspects (1) the mode of binding of the C02 substrate at the active site, and (2) the physical-chemical state of ligands on the zinc ion. 

A direct coordination of C02 to the zinc can be excluded. Based on infrared studies it was suggested that the C02 molecule is bound to the active site of carbonic 

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.  

P-lactamase Thermolysin Carboxypeptidase Alcohol dehydrogenase Alkaline phosphatase Adenosine deaminase 

FIGURE 12.2 The zinc-bound water can either be ionised to zinc-bound hydroxide, polarised by a general base to generate a nucleophile for catalysis or displaced by the substrate. 

The carbonic anhydrases of mammalian erythrocytes have been the object of extensive study for the last 66 years, and can be considered as the prototype of zinc enzymes which use the hydroxyl ion generated by ionisation of the 

FIGURE 12.3 The active site of human carbonic anhydrase. 

FIGURE 12.4 The main features of the mechanism of carbonic anhydrase. 

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Fig. 11.37 Free energy profile for the nucleophilic attack of water on CO2 (a) in aqueous solution and (b) in the enzyme carbonic anhydrase. (Graphs redrawn from Aqvist J, M Fothergill and A Warshel 1993. Computer Simulai of the COj/HCOf Interconversion Step in Human Carbonic Anhydrase I. Journal of the American Chemical Society 115 631-635.)...

Aqvist J, M FothergiU and A Warshel 1993. Computer Simulation of the CO2/HCO3 Interconversi Step in Human Carbonic Anhydrase I. Journal of the American Chemical Society 115 631-635. 

Z, ] McClarin, T Klein and R Langridge 1985. A Quantitative Structure-Activity Relationship and ecular Graphics Study of Carbonic Anhydrase Inhibitors. Molecular Pharmacology 27 493-498. 

Carbonic anhydrase red blood corpuscles carbonic acid CO, and H,0 6-8... 

Many important biochemical reactions involve Lewis acid Lewis base chemistry Carbon dioxide is rapidly converted to hydrogen carbonate ion m the presence of the enzyme carbonic anhydrase... 

Carbonic anhydrase is an enzyme that catalyzes the hydration of carbon dioxide to bicarbonate The uncatalyzed hydration of carbon dioxide is too slow to be effective m transporting carbon dioxide from the tissues to the lungs and so animals have devel oped catalysts to speed this process The activity of carbonic anhydrase is remarkable It has been estimated that one molecule of this enzyme can catalyze the hydration of 3 6 X 10 molecules of carbon dioxide per minute... 

In the chloride shift, Ck plays an important role in the transport of carbon dioxide (qv). In the plasma, CO2 is present as HCO, produced in the erythrocytes from CO2. The diffusion of HCO requires the counterdiffusion of another anion to maintain electrical neutraUty. This function is performed by Ck which readily diffuses into and out of the erythrocytes (see Fig. 5). The carbonic anhydrase-mediated Ck—HCO exchange is also important for cellular de novo fatty acid synthesis and myelination in the brain (62). 

Zinc. The 2—3 g of zinc in the human body are widely distributed in every tissue and tissue duid (90—92). About 90 wt % is in muscle and bone unusually high concentrations are in the choroid of the eye and in the prostate gland (93). Almost all of the zinc in the blood is associated with carbonic anhydrase in the erythrocytes (94). Zinc is concentrated in nucleic acids (90), and found in the nuclear, mitochondrial, and supernatant fractions of all cells. 

Metabolic Functions. Zinc is essential for the function of many enzymes, either in the active site, ie, as a nondialyzable component, of numerous metahoenzymes or as a dialyzable activator in various other enzyme systems (91,92). WeU-characterized zinc metahoenzymes are the carboxypeptidases A and B, thermolysin, neutral protease, leucine amino peptidase, carbonic anhydrase, alkaline phosphatase, aldolase (yeast), alcohol... 

Sulfonamides derived from sulfanilamide (p-arninoben2enesulfonainide) are commonly referred to as sulfa dmgs. Although several dmg classes are characterized by the presence of a sulfonamide function, eg, hypoglycemics, carbonic anhydrase inhibitors, saluretics, and tubular transport inhibitors, the antibacterial sulfonamides have become classified as the sulfa dmgs. Therapeutically active derivatives are usually substituted on the N nitrogen the position is generally unsubstituted. These features are illustrated by the stmctures of sulfanilamide (1) and sulfadiazine (2)... 

Thiadiazole-2-suifonamide, 5-acetamido-carbonic anhydrase inhibitor, 6, 576 crystal structure, 6, 548... 

FIGURE 1.19 Carbonic anhydrase, a representative enzyme, and the reaction that it catalyzes. Dissolved carbon dioxide is slowly hydrated by water to form bicarbonate ion and... 

At 20 C, the rate constant for this uncatalyzed reaction, uncat is 0.03/sec. In the presence of the enzyme carbonic anhydrase, the rate constant for this reaction, is 10 /sec. 

Thus carbonic anhydrase accelerates the rate of this reaction 3.3 X 10 times. Carbonic anhydrase is a 29-kD protein. 

Thus, the concentration of H2CO3 is itself buffered by the available pools of CO2. The hydration of CO2 is actually mediated by an enzyme, mrbonie anhydrase, which facilitates the equilibrium by rapidly catalyzing the reaction... 

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