Carbonic anhydrase Metalloenzymes

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). 

Several model systems related to metalloenzymes such as carboxypeptidase and carbonic anhydrase have been reviewed. Breslow contributed a great deal to this field. He showed how to design precise geometries of bis- or trisimidazole derivatives as in natural enzymes. He was able to synthesize a modified cyclodextrin having both a catalytic metal ion moiety and a substrate binding cavity (26). Murakami prepared a novel macrocyclic bisimidazole compound which has also a substrate binding cavity and imidazole ligands for metal ion complexation. Yet the catalytic activities of these model systems are by no means enzymic. 

Nickel is required by plants when urea is the source of nitrogen (Price and Morel, 1991). Bicarbonate uptake by cells may be limited by Zn as HCOT transport involves the zinc metal-loenzyme carbonic anhydrase (Morel et al., 1994). Cadmium is not known to be required by organisms but because it can substitute for Zn in some metalloenzymes it can promote the growth of Zn-limited phytoplankton (Price and Morel, 1990). Cobalt can also substitute for Zn but less efficiently than Cd. 

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... 

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. 

Carbonic anhydrase (carbonate dehydratase, EC 4.2.E1) is a small, monomeric zinc-containing metalloenzyme that catalyzes the reversible hydration of C02 to bicarbonate [101][102], In addition to this activity, carbonic anhydrase also catalyzes the hydrolysis of many aromatic esters [103]. 

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. 

ZnOH2 species in the zinc metalloenzyme, carbonic anhydrase (see Section 56.1.14.1.1). 

We shall now briefly outline some of the features of the zinc metalloenzymes which have attracted most research effort several reviews are available, these are indicated under the particular enzyme, and for more detailed information the reader is referred to these. Attention is focussed here, albeit briefly, on carbonic anhydrases,1241,1262,1268 carboxypeptidases, leucine amino peptidase,1241,1262 alkaline phosphatases and the RNA and DNA polymerases.1241,1262,1462 Finally, we examine alcohol dehydrogenases in rather more detail to illustrate the use of the many elegant techniques now available. These enzymes have also attracted much effort from modellers of the enzymic reaction and such studies, which reveal much interesting coordination chemistry and often new catalytic properties in their own right—and often little about the enzyme system itself (except to indicate possibilities), will be mentioned in the next section of this chapter. 

Despite the fact that carbonic anhydrase was the first zinc metalloenzyme identified1233 and a good deal is known of its structure, there is still controversy about the nature of the various active-site species and the detailed mechanisms of their action. In particular, the identity of the group with a pXa of 7 that is involved in the mechanism, and the stereochemistry around the zinc ion during catalysis, are still in dispute. The various mechanisms proposed assume either ionization of a histidine imidazole group (bound or not to the zinc) and nucleophilic attack on C02 by the coordinated imidazolate anion,1273,1274 or ionization of the Znn-coordinated water and nucleophilic attack on C02 by OH. 1271 Many papers on this problem have appeared recently and the extensive literature is the subject of the several review articles referred to above. 

Hydroxide ion coordinated to cobalt(IIl) has been observed to function as a nucleophile in both intermolecular and intramolecular reactions. The possibility that coordinated hydroxide is directly involved in the catalytic action of some metalloenzymes such as carbonic anhydrase has prompted a number of investigations of metal hydroxide reactivity towards organic substrates. Much of this chemistry has been reported and reviewed.21,23... 

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. 

In 1940 carbonic anhydrase was isolated from mammalian erythrocytes the protein was shown to contain 0.33% zinc. In 1955 carboxypeptidase became the second zinc enzyme to be reported. About 20 zinc metalloenzymes have since been studied in great detail, and about 60—70 await complete characterization. 

Carbonic anhydrase was the first known example of a zinc-containing metalloenzyme (27). It is present in a large number of tissues in all vertebrates and in many invertebrates. It has also been found in the green tissues of plants and in some bacteria (28). The primary reaction catalyzed by the enzyme... 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

It is likely that at low zinc concentrations there is impairment of the activity of vital zinc metalloenzymes such as lactic dehydrogenase, alkaline phosphatase, carbonic anhydrase, carboxypeptidase, and of enzymes in which zinc acts as a cofactor. Injection experiments showed that radioactive 65Zn preferentially concentrated in healing tissues27. ... 

In natural processes, metal ions are often in high oxidation states (2 or 3), whereas in chemical systems the metals are in low oxidation states (0 or 1). This fact inverts the role of the metal center, such that it acts as a one-electron sink in a natural system, but as a nucleophile in an artificial ones (see other chapters of this book and the review by Aresta et al. [109]). Nevertheless, important biochemical processes such as the reversible enzymatic hydration of C02, or the formation of metal carbamates, may serve as natural models for many synthetic purposes. Starting from the properties of carbonic anhydrase (a zinc metalloenzyme that performs the activation of C02), Schenk et al. proposed a review [110] of perspectives to build biomimetic chemical catalysts by means of high-level DFT or ah initio calculations for both the gas phase and in the condensed state. The fixation of C02 by Zn(II) complexes to undergo the hydration of C02 (Figure 4.17) the use of Cr, Co, or Zn complexes as catalysts for the coordination-insertion reaction of C02 with epoxides and the theoretical aspects of carbamate synthesis, especially for the formation of Mg2+ and Li+ carbamates, are discussed in the review of Schenk... 

Figure 7.6 ESI of zinc metalloenzyme carbonic anhydrase, (a)-(c) under acidic denaturing conditions (d)-(f) native state conditions and (g) native state conditions with a specific inhibitor. Structure of entry (d) is Protein Data Bank ID IBN1. Boriack-Sjodin, P.A., Zeitlin, S., Chen, H.H., Crenshaw, L, Gross S., Dantanarayana, A., Delgado, P., May J.A., Dean, T., Christianson, D.W. Structural analysis of inhibitor binding to human carbonic anhydrase II. Protein Sciv 1998, 7, 2483-2489.

A practical application for the casting approach was demonstrated in 1997 by Lehn and Hue, who developed an inhibitor for carbonic anhydrase II (a zinc-based metalloenzyme responsible for the conversion of CO2 to HC03 and H+, cf. Section 11.3.2). The process is based on the Schiff base imine forming reaction of an aldehyde and an amine, a process which is reversible under physiological... 

Substitution of foreign metals for the metals in metalloenzymes (those that contain metals as part of their structures) is an important mode of toxic action by metals. A common mechanism for cadmium toxicity is the substitution of this metal for zinc, a metal that is present in many metalloenzymes. This substitution occurs readily because of the chemical similarities between the two metals (for example, Cd2+ and Zn2+ behave alike in solution). Despite their chemical similarities, however, cadmium does not fulfill the biochemical function of zinc and a toxic effect results. Some enzymes that are affected adversely by the substitution of cadmium for zinc are adenosine triphosphate, alcohol dehydrogenase, and carbonic anhydrase. 

Carbonic anhydrase is a zinc metalloenzyme present in animals, plants and certain microorganisms which catalyses the reversible hydration of carbon dioxide and the hydration of many aldehydes. 

In our selected example, Lehn and coworkers [80] reported the synthesis of a dynamic 12-member, template-directed imine library 1, obtained from the reversible condensation of three aldehydes (monomer set M, Figure 7.11), with four primary amines (monomer set M2, Figure 7.11) in buffered aqueous conditions, followed by irreversible reduction to amines 2 with sodium cyanoborohydride. The library was prepared in the presence of a large excess of M2, to prevent further condensation of an aldehyde onto the secondary amine product. A template-driven imine library 1 was prepared in the presence of the metalloenzyme carbonic anhydrase II (CAII). After the template-assisted, reversible dynamic reaction was complete, the reducing agent was added and the amine library 2 was produced (Figure 7.11). Without any... 

Metal ions are vital to the function of many enzymes that catalyze hydrolytic reactions. Coordination of a water molecule to a metal ion alters its acid-base properties, usually making it easier to deprotonate, which can offer a ready means for catalyzing a hydrolytic reaction. Also, the placement of a metal center in the active site of a hydrolytic enzyme could permit efficient delivery of a catalytic water molecule to the hydrolyzable substrate. In fact, the first enzyme discovered, carbonic an-hydrase, is a metalloenzyme that requires a Zn2+ center for its catalytic activity (32). The function of carbonic anhydrase is to catalyze the hydrolysis of carbon dioxide to bicarbonate ... 

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