Selectivity between enzymes

It is not clear why some organisms have two 14-3-3 isoforms while others have up to 12. Binding 14-3-3 inhibits the plant enzyme nitrate reductase and there appears to be no selectivity between plant 14-3-3 isoforms in fact yeast and human isoforms appear to work equally as well in vitro. The best example where selectivity has been demonstrated is human 14-3-3o. 14-3-3o Preferential homodimerizes with itself and crystallization revealed a structural basis for this isoform s dimerization properties as well as for its specific selectivity for target binding proteins. Here partner specificity is the result of amino acid differences outside of the phosphopeptide-binding cleft. 

While there are clear differences in substrate selectivity between the drug metabolizing hydrolytic enzymes, there is also significant overlap, i.e., they will often tend to metabolize the same substrates but at different rates. For example, pseudocholinesterase, hCE-1, and hCE-2 catalyze the hydrolysis of heroin and cocaine. 

The binding sites of most enzymes and receptors are highly stereoselective in recognition and reaction with optical isomers (J, 2 ), which applies to natural substrates and synthetic drugs as well. The principle of enantiomer selectivity of enzymes and binding sites in general exists by virtue of the difference of free enthalpy in the interaction of two optical antipodes with the active site of an enzyme. As a consequence the active site by itself must be chiral because only formation of a diasteromeric association complex between substrate and active site can result in such an enthalpy difference. The building blocks of enzymes and receptors, the L-amino acid residues, therefore ultimately represent the basis of nature s enantiomer selectivity. 

Sulfamide (11) inhibits S. aureus GluRS with a. A) = 150 nmol and has only a marginal twofold selectivity between bacterial and human (HeLa GluRS, K = 300 nmol 1 ) enzymes. ... 

This approach was first applied toward an understanding of discriminating interactions in the serine proteases factor Xa, thrombin and trypsin [108] and provided selectivity information for all important serine protease subpockets, which are in agreement to experimental selectivities of typical protease inhibitors. This approach was complemented by a 3-D-QSAR selectivity analysis on a series of 3-amidinobenzyl-lH-indole-2-carboxamides [107], which points, from the viewpoint of the ligands, to similar main interactions driving selectivity between key enzymes in the blood... 

The rate of metabolic activation of l,2-dibromo-3-chloropropane in httman testicular cells is abour one-third that of rat cells. No other data are available for comparison. Nevertheless, since P450 isoenzymes and several GST enzymes are rather similar in terms of substrate selectivity between httmans and rats, it is expected that httman tissues should be capable of activating l,2-dibromo-3-chloropropane via both P450- and GST-mediated pathways. 

In many enzyme fermentations, the limiting component, usually the C-source, has to be added semi-continuously to keep its concentration at a predetermined, usually low, value. This measure makes it possible either to influence selectivity between different pathways or to uncouple predominantly cell growth during the first phase of the fermentation from predominantly product (i.e., enzyme) formation in the later stages of the fermentation cycle. Often, protein formation is induced by adding an inducer (see Chapter 4). During the fed-batch phase, the broth volume increases. Either the broth is harvested when the maximum volume is reached, or broth is withdrawn from time to time. The product is present in high concentrations. 

In the present section, analogies and similarities will be noted between enzymes and heterogeneous catalysts in the concept of the active site and metal-protein/metal-support analogies the possession of size and shape selectivity the similarity or identity in kinetics between the two processes the use of electrochemical organization on a molecular or supramolecular level the possibility of... 

Fig. 19. Schematic design of a flow injection analysis (FIA) system. A selection valve (top) allows a selection between sample stream and standard(s). The selected specimen is pumped through an injection loop. Repeatedly, the injection valve is switched for a short while so that the contents of the loop are transported by the carrier stream into the dispersion/reaction manifold. In this manifold, any type of chemical or physical reaction can be implemented (e.g. by addition of other chemicals, passing through an enzyme column, dilution by another injection, diffusion through a membrane, liquid-liquid extraction, etc. not shown). On its way through the manifold, the original plug undergoes axial dispersion which results in the typical shape of the finally detected signal peak...

An interesting attempt to overcome this problem is the design of simplified systems which try to reproduce the activity of natural enzymes (biomimetic catalysts). This approach has produced, e.g., impressive advances in the chemistry of synthetic porphyrins and in understanding the activity of some enzymes e.g. cytochrome P-450) which catalyzes oxidation reactions by an iron-porphyrin centre. Furthermore, interesting similarities have been noticed between enzymes and completely different catalysts. For instance, selective adsorption in the channels of some zeolites provide a confined, relatively hydrophobic medium even in aqueous solvent (Annex 2). This strongly resembles the active sites of several enzymes (including cytochrome P-450) that are deeply buried in hydrophobic pockets where lipophilic substrates are readily oxidized. The more hydrophilic reaction products are promptly released into... 

Enzymatic digestion has some advantages over conventional sample pretreatment procedures based on acid or alkaline digestion thus, the former uses mild conditions as regards temperature and pH, which avoids potential analyte losses by volatilization or ohemical transformation to other species, and reduces the risk of contamination. In addition, the selectivity of enzyme catalysis is a powerful tool for distinguishing between fractions of elements associated with different components of the matrix as enzymes act on oertain ohemical bonds only. 

Matrix metalloproteases (MMP) are also inhibited by hydroxamic acids and/or thiols. Over 25 variants of these enzymes are known, and some are involved in diseases ranging from inflammation to metastatic cancer (108). MMPs contain a zinc ion in the active site and function through the metallopeptidases catalytic mechanism already discussed. However, subtle differences between enzymes enable selective inhibitors to be developed (109). Fig. 15.25 lists some of the reported MMP inhibitors that use carboxylic acid (52-53), a hydroxamic acid (54-55), or thiol groups (56)as metal chelators. 

Chemicals are also used to select between different biological systems and these include agents that selectively kill one of the organisms. Common examples are the antibiotics. The antibiotics that are most useful in medicine are those that target enzymes not found in the human. An example is penicillin (Figure 8). This antibiotic resembles a substrate used for the synthesis of the cell wall of many bacteria. Penicillin will bind to the enzyme and subsequent events will inactivate the enzyme permanently. In other words, penicillin not only binds to the active sites of the bacterial enzyme, but modifies that enzyme in a manner that makes it permanently inactive. Since humans do not make cell wall, there are no enzymes of this type to be influenced and penicillin should have little effect on human enzymes. This very selective manner of killing is extremely valuable in medicine. Obviously, since the antibiotics we use are isolated from other organisms, this approach was actually developed by other life forms. 

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