Site-specific enzyme-activated

Site-specific enzyme-activated CDSs exploit specific enzymes found primarily, exclusively, or at higher activity at the site of action. 

Glycogen synthase also exists in two distinct forms which can be interconverted by the action of specific enzymes active, dephosphorylated glycogen synthase I (glucose-6-P-independent) and less active phosphorylated glycogen synthase D (glucose-6-P-dependent). The nature of phosphorylation is more complex with glycogen synthase. As many as nine serine residues on the enzyme appear to be subject to phosphorylation, each site s phosphorylation having some effect on enzyme activity. 

Since endosulfan is a cytochrome P450-dependent monooxygenase inducer, the quantification of specific enzyme activities (e.g., aminopyrine-A -demethylase, aniline hydroxylase) may indicate that exposure to endosulfan has occurred (Agarwal et al. 1978). Because numerous chemicals and drugs found at hazardous waste sites and elsewhere also induce hepatic enzymes, these measurements are nonspecific and are not necessarily an indicator solely of endosulfan exposure. However, these enzyme levels can be useful indicators of exposure, together with the detection of endosulfan isomers or the sulfate metabolite in the tissues or excreta. 

There are other types of transcriptional activators in bacteria. One is transcription factor 1 (TF1) encoded by a Bacillus subtilis phage. It is a member of the protein HU family (Chapter 27). However, unlike the nonspecific HU it binds to some sites specifically and activates transcription.143 The E. coli Ada protein is the acceptor protein in removal of methyl groups from DNA (Chapter 27). The same protein is an inducer of transcription of DNA repair enzymes in the large ada regulon. Methylation of Cys 69 of the Ada protein itself converts it into a gene activator.144... 

PT step, AGpj. This quantity is determined by the difference between the pKa s of the donor and acceptor (ApKJ. The value of this ApKa in water is the "chemical part" of the general acid-base catalysis and is independent of the specific enzyme active site. In fact, this effect can be simply considered as the result of using different reaction mechanisms with different reactants rather than an actual catalytic effect. The change of the given ApKa from its value in water to the corresponding value in the enzyme active site is a true catalytic effect. This change reflects the electrostatic effect of the enzyme active site which is the subject of the next section. 

Besides being used as adsorbent for gas molecules, both SWCNTs and MWC-NTs can be cast as a random network or a porous thin film on metal electrodes [57-59] or used as a three-dimensional scaffold [41,42] for biosensors. CNTs serve both as large immobilization matrices and as mediators to improve the electron transfer between the active enzyme site and the electrochemical transducer. Various enzymes, such as glucose oxidase and flavin adenine dinucleotide (FAD) can adsorb onto the CNT surface spontaneously and maintain their substrate-specific enzyme activity over prolonged times [57]. Recently, cells have been grown on CNT scaffolds which provide a three-dimensional permeable environment, simulating the natural extracellular matrix in a tissue [60-62]. 

The analytical sensitivity of the ELISA depends on the ability of the antibody to bind and the specific enzyme activity of the labeled immimoreactant, the conjugate. The linkage of an enzyme to an antigen or antibody may affect the specificity of an assay if any chemical modification of the moieties involved alters the antigenic determinants or the reactive sites on antibody molecules. Thus, chemical methods that do not affect these parameters have been chosen. Most of the techniques are straightforward and can be readily used by nonspecialists interested in developing their own enzyme immimoassays. 

Specificity for a particular charged substrate can be engineered into an enzyme by replacement of residues within the enzyme-active site to achieve electrostatic complementarity between the enzyme and substrate (75). Protein engineering, when coupled with detailed stmctural information, is a powerful technique that can be used to alter the catalytic activity of an enzyme in a predictable fashion. 

The a/p-barrel structure is one of the largest and most regular of all domain structures, comprising about 250 amino acids. It has so far been found in more than 20 different proteins, with completely different amino acid sequences and different functions. They are all enzymes that are modeled on this common scaffold of eight parallel p strands surrounded by eight a helices. They all have their active sites in very similar positions, at the bottom of a funnel-shaped pocket created by the loops that connect the carboxy end of the p strands with the amino end of the a helices. The specific enzymatic activity is, in each case, determined by the lengths and amino acid sequences of these loop regions which do not contribute to the stability of the fold. 

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

In recent years, biochemists have developed an arsenal of reactions that are relatively specific to the side chains of particular amino acids. These reactions can be used to identify functional amino acids at the active sites of enzymes or to label proteins with appropriate reagents for further study. Cysteine residues in proteins, for example, react with one another to form disulfide species and also react with a number of reagents, including maleimides (typically A ethylmaleimide), as shown in Figure 4.11. Cysteines also react effectively... 

Nonrepetitive but well-defined structures of this type form many important features of enzyme active sites. In some cases, a particular arrangement of coil structure providing a specific type of functional site recurs in several functionally related proteins. The peptide loop that binds iron-sulfur clusters in both ferredoxin and high potential iron protein is one example. Another is the central loop portion of the E—F hand structure that binds a calcium ion in several calcium-binding proteins, including calmodulin, carp parvalbumin, troponin C, and the intestinal calcium-binding protein. This loop, shown in Figure 6.26, connects two short a-helices. The calcium ion nestles into the pocket formed by this structure. 

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