Enzymes and catalytic activity

Living systems contain thousands of different enzymes As we have seen all are structurally quite complex and no sweeping generalizations can be made to include all aspects of enzymic catalysis The case of carboxypeptidase A illustrates one mode of enzyme action the bringing together of reactants and catalytically active functions at the active site... 

The metabolic control is exercised on certain key regulatory enzymes of a pathway called allosteric enzymes. These are enzymes whose catalytic activity is modulated through non-covalent binding of a specific metabolite at a site on the protein other than the catalytic site. Such enzymes may be allosterically inhibited by ATP or allosterically activated by ATP (some by ADP and/or AMP). 

The concept of electrostatic complimentarity is somewhat meaningless without the ability to estimate its contribution to AAg. Thus, it is quite significant that the electrostatic contribution to AAthat should be evaluated by rigorous FEP methods can be estimated with a given enzyme-substrate structure by rather simple electrostatic models (e.g., the PDLD model). It is also significant that calculated electrostatic contributions to A A g seem to account for its observed value (at least for the enzymes studied in this book). This indicates that simple calculations of electrostatic free energy can provide the correlation between structure and catalytic activity (Ref. 10). 

Aqueous solutions are not suitable solvents for esterifications and transesterifications, and these reactions are carried out in organic solvents of low polarity [9-12]. However, enzymes are surrounded by a hydration shell or bound water that is required for the retention of structure and catalytic activity [13]. Polar hydrophilic solvents such as DMF, DMSO, acetone, and alcohols (log P<0, where P is the partition coefficient between octanol and water) are incompatible and lead to rapid denaturation. Common solvents for esterifications and transesterifications include alkanes (hexane/log P=3.5), aromatics (toluene/2.5, benzene/2), haloalkanes (CHCI3/2, CH2CI2/I.4), and ethers (diisopropyl ether/1.9, terf-butylmethyl ether/ 0.94, diethyl ether/0.85). Exceptionally stable enzymes such as Candida antarctica lipase B (CAL-B) have been used in more polar solvents (tetrahydrofuran/0.49, acetonitrile/—0.33). Room-temperature ionic liquids [14—17] and supercritical fluids [18] are also good media for a wide range of biotransformations. 

This has been a brief overview of a rich field. Details of enzyme stracture and catalytic activity are studied in laboratories worldwide. Moreover, genetic engineering makes it possible to manufacture key enzymes in large quantities, so enzymes may become industrial catalysts that accomplish reactions rapidly and selectively. 

It is important to compare the catalytic properties of Prussian blue with known hydrogen peroxide transducers. Table 13.2 presents the catalytic parameters, which are of major importance for analytical chemistry selectivity and catalytic activity. It is seen that platinum, which is still considered as the universal transducer, possesses rather low catalytic activity in both H202 oxidation and reduction. Moreover, it is nearly impossible to measure hydrogen peroxide by its reduction on platinum, because the rate of oxygen reduction is ten times higher. The situation is drastically improved in case of enzyme peroxidase electrodes. However, the absolute records of both catalytic activity... 

Carbonic anhydrase is a metalloprotein with a co-ordinate bonded zinc atom immobilized at three histidine residues (His 94, His 96 and Hisl 19) close to the active site of the enzyme. The catalytic activity of the different isoenzymes varies but cytosolic CA II is notable for its very high turnover number (Kcat) of approximately 1.5 million reactions per second. 

Receptor-effector mechanisms include (1) enzymes with catalytic activities, (2) ion channels that gate the transmembrane flux of ions (ionotropic receptors), (3) G protein-coupled receptors that activate intracellular messengers (metabotropic receptors), and (4) cytosolic receptors that regulate gene transcription. Cytosolic receptors are a specific mechanism of many steroid and thyroid hormones. The ionotropic and metabotropic receptors are discussed in relevance to specific neurotransmitters in chapter 2. 

Another possible explanation for the limitations of catalytic antibodies raised against TSA can be found in the different accessibility of the active site. In the case of natural enzymes, it is that their catalytic machinery and bound substrates are often buried. This feature isolates from the solvent the reactive functionalities that mediate chemical transformations. On the contrary, in antibody catalysis, the moieties of the bound haptens that mimic the TS are often positioned near the entrance of the antibody-combining site. This disparity in the overall architecture of natural enzymes and catalytic antibodies is undoubtedly a factor in the lower catalytic... 

Efforts should be made to stabilize an enzyme s activity. Certain agents (such as glycerol, ammonium ions, boric acid, polyethylene glycol, and even talcum powder or bentonite clay) have proven widely to be effective enzyme stabilizers. For multisubstrate enzymes, inclusion of one particular substrate with the enzyme (in the absence of other substrates or cofactors) often stabilizes an enzyme s catalytic activity. Such a substrate may also assist in unlocking the enzyme from a particularly inactive conformational form. In addition to substrates, other ligands and effectors (including reaction products. 

It is possible to attach photochromic molecules onto naturally occurring receptors and enzymes and by so doing be able to photoregulate their binding and catalytic activities. These materials have the potential to be used as chemotherapeutic agents and biosensors, and as bioelectronic materials. In most of this work to date spiropyrans have been used as the photochromic element in the system. 

In concert, structure determinations and enzymological studies for catalytic rates and product distributions with structurally varied aldehydes of native enzymes and numerous active-site mutants have allowed us to derive a conclusive blueprint for the catalytic cycle of FucA (Fig. 2.2.5.2). The proposed mechanism, which has general implications for other metal-dependent aldolases, is able to rationalize all key stereochemical issues successfully ]15]. Independent work by other groups has recently provided further insight into related proteins with Fru A and TagA specificity [16]. 

This interesting result strongly suggests that even for the substituted cyclodextrins the capacity of inclusion formation is much the same as for the parent cyclodextrin. Therefore, we may extend the basic concept of the structure of cyclodextrin inclusion to molecular design for the preparation of artificial enzymes having satisfactory substrate specificities and catalytic activities. 

Enzyme flexibility is greater in solvents with high polarity because of weaker electrostatic interactions in these solvents [54, 104, 105]. The loss in enzyme activity seen in the NMR study described above may be attributed to the water stripping model as water is stripped from the enzyme, locations in and on the enzyme previously inaccessible to the solvent may become accessible, thus permitting increased solvent-enzyme interactions [103]. As a result, enzyme structure may be disrupted (e.g., partially denatured), and catalytic activity is decreased. The partially denatured enzyme appears to exhibit greater flexibility as solvent polarity increases [106, 107]. 

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