Carbonic anhydrase enzyme efficiency

A general definition of the Quantum Molecular Similarity Measure is reported. Particular cases of this definition are discussed, drawing special attention to the new definition of Gravitational-like Quantum Molecular Similarity Measures. Applications to the study of fluoromethanes and chloro-methanes, the Carbonic Anhydrase enzyme, and the Hammond postulate are presented. Our calculations fully support the use of Quantum Molecular Similarity Measums as an efficient molecular engineering tool in order to predict physical properties, lMok>gical and pbarraacdogical activities, as well as to interpret complex chemical problems. 

The most fundamental process dealing with the activation of C02 involves the hydration of C02 to produce bicarbonate and the reverse dehydration of bicarbonate to produce C02. These processes are of biological and environmental significance since they control the transport and equilibrium behavior of C02. The spontaneous hydration of C02 and dehydration of HCO3 are processes that are too slow and must therefore be catalyzed by metal complexes in order to expedite the overall conversion rate. In biological systems, a series of enzymes, the carbonic anhydrases, are the efficient catalysts and can accelerate the reactions by up to 7 orders of magnitude. The mechanism of this... 

Importantly, carbonic anhydrase II is one of the most efficient biological catalysts known and it catalyzes the hydration of CO2 with a turnover rate of 10 sec at 25 C (Khalifah, 1971 Steiner et al, 1975). With kcaJKm = 1.5 X 10 sec carbonic anhydrase II is one of a handful of enzymes for which catalysis apparently approaches the limit of diffusion control. Since transfer of the product proton away from the enzyme to bulk solvent comprises a kinetic obstacle [an enzyme-bound group with ap/C, of about 7 cannot transfer a proton to bulk solvent at a rate faster than 10 sec (for a review see Eigen and Hammes, 1963)], the observed turnover rate of 10 sec" requires the participation of buffer in the proton transfer. 

Compounds which enhance the catalytic activities of the CAs are known as activators. Activators of carbonic anhydrases are less studied because CA is one of the most efficient enzymes known. Carbonic anhydrase II activation by phosphorylation in the presence of protein kinase and cAMP has been reported (195,196). Also some anions are activators for CA III (197,198) the catalytic effect is due to the proton shuttling capacities of such activators. Histamine, a well known activator, for native and Co(II)-substituted isoenzymes I and II CA is reported by Briganti et al. (199). Amines [Ar-CH(R3)CH(R2)NH(R1) Ar =Aromatic/heterocyclic group R1 =R2 = H, Me R3 = H, OH, COOH] and amino acids are efficient activators for CA I—III (200-207). These amines possess a bulky aromatic/heterocyclic moiety in their molecular structure and act as proton acceptor (204-207). 

Examples of other recombinant enzymes in which an alteration using site-directed mutagenesis resulted in altered substrate binding efficiencies, rates of catalysis, or stability include carbonic anhydrase (Alexander, Nair Christianson, 1991), lactate dehydrogenase (Feeney, Clarke Holbrook, 1990), and several industrially important proteases (Wells etal., 1987 Siezenera/., 1991 Teplyakovcra/., 1992 Aehle et al., 1993 Rheinnecker et al., 1994). 

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. 

Some other reactions, such as aldehyde hydration (29) and e ter hydrolyses (30—33) are also catalyzed by the enzyme, but much j ess efficiently than the reversible hydration of CO 2. The esterase reaction, in particular, has been very useful in the kinetic analysis of carbonic anhydrase function, however. 

General acid-base catalysis is often the controlling factor in many mechanisms and acts via highly efficient and sometimes intricate proton transfers. Whereas log K versus pH profiles for conventional acid-base catalyzed chemical processes pass through a minimum around pH 7.0, this pH value for enzyme reactions is often the maximum. In enzymes, the transition metal ion Zn2+ usually displays the classic role of a Lewis acid, however, metal-free examples such as lysozyme are known too. Good examples of acid-base catalysis are the mechanisms of carbonic anhydrase II and both heme- and vanadium-containing haloperoxidase. 

Carbonic anhydrase II, present in human red blood cells (RBCs), catalyzes the reversible hydration of C02. It is one of the most efficient enzymes and only diffusion-limited in its turnover numbers. The catalytic Zn11 is ligated by three histidine residues and OH this ZnOH+ structure renders the zinc center an efficient nucleophile which is able to attack the C02 molecule and capture it in an adjacent hydrophobic pocket. The catalytic mechanism is shown in Figure 9.5. 

The study of zinc-aqua complexes as synthetic carbonic anhydrase models has shown a low coordination number and a hydrophobic environment to be prerequisites for a low pK value of the aqua ligand, which is essential for efficient enzyme function. The pK value in the case of 13 is expected to be greater than 10.7, which is the value determined for pentacoordinate [Zn(tren)(H20)](C104)2, in which the water ligand is more strongly bound and thus expected to be more acidic (cf. the discussion of bond lengths, above). Thus, 13 is not expected to show carbonic anhydrase-like reactivity. However, in related tetraazamacrocyclic systems it has been shown that upon... 

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 ... 

This crucial reaction allows the carbon dioxide that forms in the cells during metabolism to be removed. If the carbon dioxide were allowed to accumulate, it would poison the cell. Carbonic anhydrase is so efficient that one molecule of enzyme can catalyze the reaction of over 600,000 carbon dioxide molecules in one second ... 

Because zinc is required for the growth of phytoplankton, its availability affects the biological pump. Although zinc limitation of an entire phytoplankton community has never been demonstrated, levels of dissolved zinc are often low enough to limit many taxa (Morel et al, 1994 Sunda and Huntsman, 1995b Timmermans et al, 2001). Zinc is an integral part of the enzyme, carbonic anhydrase (Morel et al, 1994), which helps maintain an efficient supply of CO2 to Rubisco. 

The enzyme carbonic anhydrase allows this reaction to take place 10 million times faster than it normally would. The forward and reverse processes are accelerated equally. Hence the reaction s equilibrium constant is unaffected by the enzyme s presence. Enzymes are very efficient. A single molecule of carbonic anhydrase can cause 600 000 carbon dioxide molecules to react each second. 

It is of interest to note that the efficient enzymes, catalase, carbonic anhydrase and the nitrogenases have very small substrates (HjOj, CO2 and Nj, respectively) for which it is not easy to distinguish between reacting and non-reacting parts. It is a great challenge to quantitatively understand the forces of interaction between these substrates and their enzymes. 

Much has been done, said, and written in enzyme kinetics and I will mention only a few things. The enzymes are usually selective they catalyze only a single reaction or only one type of reaction. While enzymes generally speed up the reactions, in comparison to the same reaction conducted in the laboratory, the enzymes that are not very selective are usually relatively slow. On the other hand, certain highly selective enzymes, like carbonic anhydrase or glutamate mutase, can speed up the reaction conducted under laboratory conditions by a factor of 10 -lO, that is, trillion-to quadrillion-fold. No man-made catalyst matches this efficiency. Thousands of enzymes are known today they are catalogued into six major categories, in relation to the type of chemical reaction they catalyze. Each enzyme is identified by its enzyme code number, or E.C. number [5]. 

In parallel work, the synthetic enzyme has been gradually refined through further optimization. In a later paper [139], human carbonic anhydrase 11 was combined with a sulfonamide derived from pyridine/IrCp to create a catalyst for the ATH of imines. The X-ray structure of a derivative was used to improve the catalytic performance, ultimately achieving 68 % e.e. A ribonuclease has also been used as the basis of an artificial amine reductase. In this case, the incorporation of a Cp lr complex gave an efficient and selective artificial enzyme for imine reduction [140]. 

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