Metalloenzymes reversal

The addition of HCN to aldehydes or ketones produces cyanohydrins (a-hydroxy nitriles). Cyanohydrins racemize under basic conditions through reversible loss of FiCN as illustrated in Figure 6.30. Enantiopure a-hydroxy acids can be obtained via the DKR of racemic cyanohydrins in the presence of an enantioselective nitriletransforming enzyme [86-88]. Many nitrile hydratases are metalloenzymes sensitive to cyanide and a nitrilase is usually used in this biotransformation. The DKR of mandelonitrile has been extended to an industrial process for the manufacture of (R)-mandelic acid [89]. 

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]. 

Researchers studying the metalloenzyme hydrogenase would like to design small compounds that mimic this enzyme s ability to reversibly reduce protons to H2 and H2 to 2H+, using an active center that contains iron and nickel. Cobalamins (vitamin and its derivatives) contain an easily activated Co-C bond that has a number of biological functions, one of which is as a methyl transferase, 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR). This enzyme converts homocysteine (an amino acid that has one more CH2 group in its alkyl side chain than cysteine see Figure 2.2) to methionine as methylcobalamin is converted to cobalamin. 

This zinc metalloenzyme [EC 1.1.1.1 and EC 1.1.1.2] catalyzes the reversible oxidation of a broad spectrum of alcohol substrates and reduction of aldehyde substrates, usually with NAD+ as a coenzyme. The yeast and horse liver enzymes are probably the most extensively characterized oxidoreductases with respect to the reaction mechanism. Only one of two zinc ions is catalytically important, and the general mechanistic properties of the yeast and liver enzymes are similar, but not identical. Alcohol dehydrogenase can be regarded as a model enzyme system for the exploration of hydrogen kinetic isotope effects. 

The problem of antibiotic-resistant bacteria has created a pressing demand for new antibacterial agents with novel mechanisms of action. Af-Formylhydroxylamines, also known as reverse- or retro-hydroxamates, are one of the few novel targets that are currently being pursued against a variety of metalloenzyme targets, including carboxy... 

Titration with chelators of a metalloenzyme preparation from which extraneous metals and chelators have been removed produces a characteristic enhancement of the intrinsic protein fluorescence (excitation at 280 nm, emission at 350 nm) (13). This fluorescence enhancement by nonfluorescent chelators is instantaneous, reversible by excess added divalent metal ions, and can occur without loss of activity. Different chelators give different characteristic amounts of fluorescence enhancement at saturation, demonstrating the specific effect of the chelator on the fluorescence of the apparent metalloenzyme-chelator complex. In contrast, if the effect of chelators were simply to complex with Mg2 after its dissociation from the metalloenzyme, the resulting apoenzyme should have identical fluorescence properties regardless of which chelator was utilized. 

Metalloenzyme is represented by E(Mg2 ), C is chelator, E(Mg2 C) represents chelator reversibly bound to the metalloenzyme with dissociation constant KD and with characteristic enhancement of the protein fluorescence, and k is the first-order rate constant for irreversible formation of inactive protein P and the cation-chelator complex, Mg2+C. Values of both Kd and k may vary according to the nature of the chelator, the nature of the metal ion (if other cations can replace Mg2 ), and the intrinsic stability of the cation-chelator complex, as well as pH, temperature, and protein concentration. 

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. 

The alcohol dehydrogenases are zinc metalloenzymes which can oxidize a wide variety of alcohols to their corresponding aldehydes or ketones using nicotinamide adenine dinucleotide (NAD+) as coenzyme. These reactions are readily reversible so that carbonyl compounds may be reduced by NADH. 

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... 

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... 

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... 

In addition to catalyzing the oxidative or reductive transformation of small molecules, redox metalloenzymes can also effect the translocation of protons against a chemical gradient across a membrane. Conceptually, this movement can be understood from the point of view of the fundamental coordination chemistry of metal-oxo complexes (46, 47). Reversible reduction of metal-oxo complexes often occurs concomitantly with protonation, as shown in equations 7 and 8. 

Investigations into the metalloenzyme nature of an enzyme frequently involves an attempt to inhibit it with a chelator, followed by reversal of any inhibition by adding excess metal. If excess zinc doesn t reverse the inhibition, it may be due to the fact that zinc can inhibit the zinc enzyme. A considerable amount of anecdotal information exists that zinc inhibits zinc enzymes but how it accomplishes it is not well established. The most thorough study of its mechanism of inhibition is on CPD... 

The accuracy of the microwave-excitation spectrometric method was verified by comparing results from it with those of atomic absorption analysis for readily available metalloenzymes of known zinc stoichiometry. Carboxypeptidase A (EC 3.4.12.2), carbonic anhydrase (EC 4.2.1.1), alcohol dehydrogenase (EC l.l.l.l), and alkaline phosphatase (EC 3.1.3.1) were dialyzed vs. metal-free bufiFers, then diluted with 10 mmol/ L KCl or 1 mmol/L HCl for metal analysis (24). For atomic absorption analysis, at least lOO- xg samples were required, but microwave excitation required only 0.1 fig. Even though 1000-fold less protein was required for microwave excitation analysis, the agreement between the data obtained by the two methods is excellent (Table II). So little of the reverse transcriptase was available to us that we could not use atomic absorption for its analyses. 

Reductions of metalloenzyme activity induced by the deficiency may be partially or whoUy restored by effective treatment. Reversal of hematological and immune function laboratory abnormahties can also be used, as can hormonal changes induced by the deficiency. 

Thus, red blood cell carbonic anhydrase which catalyzes the reversible hydration of CO2, plays a vital role in carbon dioxide transport and elimination. Carbonic anhydrase is a monomeric (M.W. 29,000) zinc metalloenzyme and is... 

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