Zinc enzyme, carbonic anhydrase

This is of relevance to the mechanism of carbonic anhydrase. This enzyme, which catalyzes the hydration of C02, has at its active site a Zn2+ ion ligated to the imidazole rings of three of its histidines. The classic mechanism for the reaction is that the fourth ligand is a water molecule which ionizes with a pKa of 7.37 The reactive species is considered to be the zinc-bound hydroxyl. Chemical studies show that zinc-bound hydroxyls are no exception to the rule of high reactivity. The H20 in structure 2.31 ionizes with a pKa of 8.7 and catalyzes the hydration of carbon dioxide and acetaldehyde.38... 

Carbonic anhydrase is a zinc(II) metalloenzyme which catalyzes the hydration and dehydration of carbon dioxide, C02+H20 H+ + HC03. 25 As a result there has been considerable interest in the metal ion-promoted hydration of carbonyl substrates as potential model systems for the enzyme. For example, Pocker and Meany519 studied the reversible hydration of 2- and 4-pyridinecarbaldehyde by carbonic anhydrase, zinc(II), cobalt(II), H20 and OH. The catalytic efficiency of bovine carbonic anhydrase is ca. 108 times greater than that of water for hydration of both 2- and 4-pyridinecarbaldehydes. Zinc(II) and cobalt(II) are ca. 107 times more effective than water for the hydration of 2-pyridinecarbaldehyde, but are much less effective with 4-pyridinecarbaldehyde. Presumably in the case of 2-pyridinecarbaldehyde complexes of type (166) are formed in solution. Polarization of the carbonyl group by the metal ion assists nucleophilic attack by water or hydroxide ion. Further studies of this reaction have been made,520,521 but the mechanistic details of the catalysis are unclear. Metal-bound nucleophiles (M—OH or M—OH2) could, for example, be involved in the catalysis. 

Zinc is the second most important of the essential trace elements for humans. It is a constituent of some enzymes, such as carbonic anhydrase. Zinc is sufficiently abundant that deficiencies of zinc are unknown. The highest levels of zinc are found in shellfish, which may contain 400 ppm. The level of zinc in cereal grains is 30 to 40 ppm. When acid foods such as fruit juices are stored in galvanized containers, sufficient zinc may be dissolved to cause zinc poisoning. The zinc in meat is tightly bound to the myofibrils and has been speculated to influence meat s water-binding capacity (Hamm 1972). 

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. 

Proximal tubular epithelial cells are richly endowed with the zinc metalloenzyme carbonic anhydrase, which is fonnd in the luminal and basolateral membranes (type IV carbonic anhydrase, an enzyme tethered to the membrane by a glycosylphosphatidylinositol linkage), as well as in the cytoplasm (type 11 carbonic anhydrase). Carbonic anhydrase plays a key role in NaHCOj reabsorption and acid secretion. 

Currently, D.W. Christianson (University of Pennsylvania, Philadelphia, Pennsylvania) and C.A. Fierke (Duke University, Durham, North Carolina) are looking at redesigning the zinc binding site of the human carbonic anhydrase II enzyme and E. Kimura s group (Hiroshima University, Hiroshima, Japan) is examining the roles of zinc(ll) ion in zinc enzymes. 

Recall from the "Chemistiy and Life" box in Section 2.7 that zinc is needed in our diets in trace amounts. Zinc is an essential part of several enzymes, the proteins that facilitate or regulate the speeds of key biological reactions. For example, one of the most important zinc-containing enzymes is carbonic anhydrase. This enzyme is found in red blood cells. Its job is to facilitate the reaction of carbon dioxide (CO2) with water to form the bicarbonate ion (HCOs ) ... 

An enzyme of which a metal (ion) is an integral part of its active structure. Examples carbonic anhydrase (zinc) and cytochromes (iron, copper). 

Carbonic Anhydrase. One of the first proteins studied by halogen NMR, where an artificial metal label is not used, was carbonic anhydrase. This enzyme has a molecular weight of about 30,000 and contains one zinc atom at, or near, the active center. The enzyme has great physiological significance primarily since it catalyses the reversible hydration of carbon dioxide (eq. 28). 

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... 

Zinc, see also Enzyme cofactors as cofactor for alcohol dehydrogenase, 205 as cofactor for carbonic anhydrase, 197-200... 

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... 

Mn(II)-substituted carbonic anhydrase, which is less active than the zinc enzyme, has also been postulated to exhibit bidentate bicarbonate coordination. 

Carbonic anhydrase (CA) exists in three known soluble forms in humans. All three isozymes (CA I, CA II, and CA III) are monomeric, zinc metalloenzymes with a molecular weight of approximately 29,000. The enzymes catalyze the reaction for the reversible hydration of C02. The CA I deficiency is known to cause renal tubular acidosis and nerve deafness. Deficiency of CA II produces osteopetrosis, renal tubular acidosis, and cerebral calcification. More than 40 CA II-defi-cient patients with a wide variety of ethnic origins have been reported. Both syndromes are autosomal recessive disorders. Enzymatic confirmation can be made by quantitating the CA I and CA II levels in red blood cells. Normally, CA I and CAII each contribute about 50% of the total activity, and the CAI activity is completely abolished by the addition of sodium iodide in the assay system (S22). The cDNA and genomic DNA for human CA I and II have been isolated and sequenced (B34, M33, V9). Structural gene mutations, such as missense mutation, nonsense... 

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. 

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... 

There has been particular recent interest in zinc nitrate complexes as coordination models for bicarbonate binding in carbonic anhydrase. The mono- or bidentate coordination modes have been studied with tris-pyrazolyl borate complexes and can be rationalized in the context of the enzyme activity.433 Caution in this comparison is introduced by ab initio calculations on these model systems demonstrating both monodentate and bidentate coordination energy minima for nitrate binding to zinc 434... 

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... 

Synthesis of functional models of carbonic anhydrase has been attempted with the isolation of an initial mononuclear zinc hydroxide complex with the ligand hydrotris(3-t-butyl-5-methyl-pyrazolyl)borate. Vahrenkamp and co-workers demonstrate the functional as well as the structural analogy to the enzyme carbonic anhydrase. A reversible uptake of carbon dioxide was observed, although the unstable bicarbonate complex rapidly forms a dinuclear bridged complex. In addition, coordinated carbonate esters have been formed and hydrolyzed, and inhibition by small ions noted.462 A number of related complexes are discussed in the earlier Section 6.8.4. 

The importance of zinc may extend even further as the processes that control carbon uptake by marine phytoplankton are important in the carbon cycle, and the zinc enzyme carbonic anhydrase could be a limiting factor. The concentration of zinc available to some marine phytoplankton has... 

Theoretical calculations have been carried out on a number of zinc-containing enzymatic systems. For example, calculations on the mechanism of the Cu/Zn enzyme show the importance of the full protein environment to get an accurate description of the copper redox process, i.e., including the electronic effects of the zinc ion.989 Transition structures at the active site of carbonic anhydrase have been the subject of ab initio calculations, in particular [ZnOHC02]+, [ZnHC03H20]+, and [Zn(NH3)3HC03]+.990... 

Stanton and Merz studied the reaction of carbon dioxide addition to zinc hydroxide, as a model for zinc metallo-enzyme human carbonic anhydrase IIJ 36. It was shown that the LDA calculations (DFT(SVWN)) were not reliable for locating transition state structures whereas the post-LDA ones (DFT(B88/P86)) led to the transition state structures and ener-... 

The first recognition of a role for zinc in biochemistry was the discovery inl939 that it was an essential component of carbonic anhydrase, a key enzyme in erythrocytes that catalyses the formation of bicarbonate ... 

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... 

L-amino acids as effident activators of zinc enzyme carbonic anhydrase. Liebigs Annalen der Chemie,... 

Although zinc, cadmium, and mercury are not members of the so-called main-group elements, their behavior is very similar because of their having complete d orbitals that are not normally used in bonding. By having the filled s orbital outside the closed d shell, they resemble the group IIA elements. Zinc is an essential trace element that plays a role in the function of carboxypeptidase A and carbonic anhydrase enzymes. The first of these enzymes is a catalyst for the hydrolysis of proteins, whereas the second is a catalyst for the equilibrium involving carbon dioxide and carbonate,... 

One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

The system illustrated by (272) forms the basis of a model for the zinc-containing metalloenzyme, carbonic anhydrase (Tabushi Kuroda, 1984). It contains Zn(n) bound to imidazole groups at the end of a hydrophobic pocket, as well as basic (amine) groups which are favourably placed to interact with a substrate carbon dioxide molecule. These are both features for the natural enzyme whose function is to catalyze the reversible hydration of carbon dioxide. The synthetic system is able to mimic the action of the enzyme (although side reactions also occur). Nevertheless, the formation of bicarbonate is still many orders of magnitude slower than occurs for the enzyme. 

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