Water carbonic anhydrase

Carbonic anhydrase is an enzyme that produces free hydrogen ions, which are then exchanged for sodium ions in the kidney tubules. Carbonic anhydrase inhibitors inhibit the action of the enzyme carbonic anhydrase This effect results in the excretion of sodium, potassium, bicarbonate, and water. Carbonic anhydrase inhibitors also decrease the production of aqueous humor in the eye, which in turn decreases intraocular pressure (IOP) (ie, the pressure within the eye). 

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.)...

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... 

Among one of the more unusual side effects noticed as the use of the sulfonamides became widespread was the increased urine output of many patients treated with these drugs. The fact that the urine was unusually alkaline led to the suspicion, later (Confirmed by independent means, that these agents were responsible for partial inhibition of the enzyme carbonic anhydrase. Inhibition of this enzyme causes increased excretion of sodium and bicarbonate ions as well as water, in effect bringing about diure-... 

Micelles in water are described as spherical aggregates of a surfactant monomer27 30). They somewhat resemble to enzyme proteins in structures and functions, although the details are yet the subjects of recent controversies 29,30). There are numerous studies of micellar models of enzymes 28), but the examples of those of metalloenzymes are very few 31 37). In particular, there are no examples of micellar models of carboxypeptidase or carbonic anhydrase except ours 36,37). 

FIGURE 8.8. Calculated free-energy profile for the reaction of carbonic anhydrase. g2(a) and g2(b) designate the states where the proton acceptors are water and histidine respectively. 

Figure 6-9. The Bohr effect. Carbon dioxide generated in peripheral tissues combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. Deoxyhemoglobin acts as a buffer by binding protons and delivering them to the lungs. In the lungs, the uptake of oxygen by hemoglobin releases protons that combine with bicarbonate ion, forming carbonic acid, which when dehydrated by carbonic anhydrase becomes carbon dioxide, which then is exhaled.

X-ray diffraction studies on several forms of the enzyme have demonstrated that the active site is composed of a pseudo-tetrahedral zinc center coordinated to three histidine imidazole groups and either a water molecule [(His)3Zn-OH2]2+ (His = histidine), or a hydroxide anion [(His)3Zn-OH] +, depending upon pH (156,157). On the basis of mechanistic studies, a number of details of the catalytic cycle for carbonic anhydrase have been elucidated, as summarized in Scheme 22... 

The value of the tris(pyrazolyl)hydroborato complexes [TpRR ]ZnOH is that they are rare examples of monomeric four-coordinate zinc complexes with a terminal hydroxide funtionality. Indeed, [TpBut]ZnOH is the first structurally characterized monomeric terminal hydroxide complex of zinc (149). As such, the monomeric zinc hydroxide complexes [TpRR ]ZnOH may be expected to play valuable roles as both structural and functional models for the active site of carbonic anhydrase. Although a limitation of the [TpRR ]ZnOH system resides with their poor solubility in water, studies on these complexes in organic solvents... 

Carbonic anhydrase inhibitors decrease aqueous humor production by inhibition of the carbonic anhydrase isoenzyme II located in the ciliary body. In the eye, carbonic anhydrase catalyzes the conversion of water and carbon dioxide to bicarbonate and hydrogen ion, which is a significant step in aqueous humor production. Carbonic anhydrase inhibitors are available in systemic and topical preparations.10,13,14... 

When carbonic acid levels become too high, the body releases an enzyme called carbonic anhydrase, which catalyzes the breakdown of carbonic acid into carbon dioxide and water ... 

As an illustration, we briefly discuss the SCC-DFTB/MM simulations of carbonic anhydrase II (CAII), which is a zinc-enzyme that catalyzes the interconversion of CO2 and HCO [86], The rate-limiting step of the catalytic cycle is a proton transfer between a zinc-bound water/hydroxide and the neutral/protonated His64 residue close to the protein/solvent interface. Since this proton transfer spans at least 8-10 A depending on the orientation of the His 64 sidechain ( in vs. out , both observed in the X-ray study [87]), the transfer is believed to be mediated by the water molecules in the active site (see Figure 7-1). To carry out meaningful simulations for the proton transfer in CAII, therefore, it is crucial to be able to describe the water structure in the active site and the sidechain flexibility of His 64 in a satisfactory manner. 

CO3 species was formed and the X-ray structure solved. It is thought that the carbonate species forms on reaction with water, which was problematic in the selected strategy, as water was produced in the formation of the dialkyl carbonates. Other problems included compound solubility and the stability of the monoalkyl carbonate complex. Van Eldik and co-workers also carried out a detailed kinetic study of the hydration of carbon dioxide and the dehydration of bicarbonate both in the presence and absence of the zinc complex of 1,5,9-triazacyclododecane (12[ane]N3). The zinc hydroxo form is shown to catalyze the hydration reaction and only the aquo complex catalyzes the dehydration of bicarbonate. Kinetic data including second order rate constants were discussed in reference to other model systems and the enzyme carbonic anhy-drase.459 The zinc complex of the tetraamine 1,4,7,10-tetraazacyclododecane (cyclen) was also studied as a catalyst for these reactions in aqueous solution and comparison of activity suggests formation of a bidentate bicarbonate intermediate inhibits the catalytic activity. Van Eldik concludes that a unidentate bicarbonate intermediate is most likely to the active species in the enzyme carbonic anhydrase.460... 

The carbon dioxide produced during cellular metabolism diffuses out of the cells and into the plasma. It then continues to diffuse down its concentration gradient into the red blood cells. Within these cells, the enzyme carbonic anhydrase (CA) facilitates combination of carbon dioxide and water to form carbonic acid (H2C03). The carbonic acid then dissociates into hydrogen ion (H+) and bicarbonate ion (HC03). 

FIGURE 6.7 A HILIC column (Tosoh Amide-80, 25 cm x 4.6 mm, 5 lm particles) at different flow rates with a solvent of ACN/water/0.1%TFA. The gradient is 80% to 15% ACN. Protein standards A = aprotinin, B = cytochrome C, C = carbonic anhydrase. 

As an example, consider an early calculation of isotope effects on enzyme kinetics by Hwang and Warshel [31]. This study examines isotope effects on the catalytic reaction of carbonic anhydrase. The expected rate-limiting step is a proton transfer reaction from a zinc-bound water molecule to a neighboring water. The TST expression for the rate constant k is... 

This reaction is essential in maintaining a constant pH in blood by the bicarbonate buffer system. Carbonic anhydrase, which contains a single zinc atom in its structure, has a molecular weight of about 30,000. In this structure, zinc is surrounded tetrahedrally by three histidine molecules and one water molecule. The exact role of the catalyst is not known, but it is believed to involve hydrolysis that can be represented as... 

In turn, the concentration of C02 in the atmosphere depends on the mass of the biosphere and its rate of decay after death, and on the carbonic-anhydrase concentrations in the sea surface. In future predictions of the rate of increase of C02 partial pressure in the atmosphere due to burning fossil fuels, it will be important to include the interaction of the atmospheric C02 with the bio-organic reservoir and the catalyzation of its absorption into the sea by means of the action of carbonic-anhydrase dissolved in sea water, considerations which have not been taken into account in past computations. 

Figure 2. Sodium and chloride uptake across an idealised freshwater-adapted gill epithelium (chloride cell), which has the typical characteristics of ion-transporting epithelia in eukaryotes. In the example, the abundance of fixed negative charges (muco-proteins) in the unstirred layer may generate a Donnan potential (mucus positive with respect to the water) which is a major part of the net transepithelial potential (serosal positive with respect to water). Mucus also contains carbonic anhydrase (CA) which facilitates dissipation of the [H+] and [HCO(] to CO2, thus maintaining the concentration gradients for these counter ions which partly contribute to Na+ import (secondary transport), whilst the main driving force is derived from the electrogenic sodium pump (see the text for details). Large arrow indicates water flow...

The first zinc enzyme to be discovered was carbonic anhydrase in 1940, followed by car-boxypeptidase A some 14 years later. They both represent the archetype of mono-zinc enzymes, with a central catalytically active Zn2+ atom bound to three protein ligands, and the fourth site occupied by a water molecule. Yet, despite the overall similarity of catalytic zinc sites with regard to their common tetrahedral [(XYZ)Zn2+-OH2] structure, these mononuclear zinc enzymes catalyse a wide variety of reactions, as pointed out above. The mechanism of action of the majority of zinc enzymes centres around the zinc-bound water molecule,... 

Fig. 17. Rapid reversed-phase separation of proteins at a flow-rate of 10 ml/min (Reprinted with permission from [127]. Copyright 1999 Elsevier). Conditions Column, 50x4.6 mm i.d. poly(styrene-co-divinylbenzene) monolith,mobile phase gradient 42% to 90% acetonitrile in water with 0.15% trifluoroacetic acid in 0.35 min, UV detection at 280 nm. Peaks ribonucle-ase (1), cytochrome c (2), bovine serum albumin (3), carbonic anhydrase (4), chicken egg albumin (5)...

In the other subdivision, water activation occurs in the first step of the enzymatic cycle. This activation is achieved by a carboxylate group in aspartic hydrolases (Fig. 3.10), Zn2+ and a carboxy group in metallopep-tidases (Fig. 3.12 ), a histidine side chain in calcium-dependent hydrolases (Fig. 3.14), or a Zn2+ in carbonic anhydrase (Fig. 3.15). The substrate, on the other hand, is polarized (activated) by a carboxy group in aspartic hydrolases or by a cation in metallopeptidases and calcium-dependent hydrolases. In this manner, the reactivity of both the water molecule and the substrate is enhanced and fine-tuned to drive formation of a tetrahedral intermediate that will break down to form the hydrolysis products. 

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