Enzymes attachment

Generally inhibitors are competitive or non-competitive with substrates. In competitive inhibition, the interaction of the enzyme with the substrate and competitive inhibitor instead of the substrate can be analysed with the sequence of reactions taking place as a result, a complex of the enzyme-inhibitor (El) is formed. The reaction sets at equilibrium and the final step shows the product is formed. The enzyme must get free, but the enzyme attached to the inhibitor does not have any chance to dissociate from the El complex. The El formed is not available for conversion of substrate free enzymes are responsible for that conversion. The presence of inhibitor can cause the reaction rate to be slower than the ordinary reaction, in the absence of the inhibitor. The sequence of reaction mechanisms is ... 

A great savings in enzyme consumption can be achieved by immobilizing the enzyme in the reactor (Fig. 12). In addition to the smaller amount of enzyme required, immobilization often increases the stability of the enzyme. Several designs of immobiliz-ed-enzyme reactors (lERs) have been reported, with open-tubular and packed-bed being the most popular. Open-tubular reactors offer low dispersion but have a relatively small surface area for enzyme attachment. Packed-bed reactors provide extremely high surface areas and improved mass transport at the cost of more dispersion. 

DNA polymerase 1. For most applications DNA polymerase 1 from E. coli is used. The enzyme attaches to a short single-stranded region in a dsDNA molecule and then synthesizes a new strand of DNA, degrading the existing strand as it proceeds. When used in vitro this incubation is carried out at 12-15 °C to prevent more than one round of replication occurring. It is used for in vitro labelling of DNA by the nick translation method (described below). 

This complex results in catalysis. The remainder of the amino acids in the enzyme are involved in maintenance of the three-dimensional struc-tnre of the enzyme, attaching the enzyme molecule to intracellular structures (e.g. membranes) or in binding molecules that regulate the activity of the enzyme. An introdnction to the chemistry involved in reactions, and the bonds that are involved in the structures of small and large molecnles is provided is Appendix 3.1. This shonld help students with material in this and other chapters. 

The covalent linking of enzymes to supports is mainly of use in cases where stabile and strong enzyme attachment is important. However, coupling to amino acids residues responsible for the catalytic activity should be avoided to preserve the active site. The carrier can be activated using a number of chemical approaches depending on the nature of the carrier e.g. Sepharose or CPG and the enzyme of interest. This field has been investigated thoroughly, for reviews see references. 

Disaccharides must be hydrolyzed to monosaccharides before entering cells. Intestinal disaccharides and dextrins are hydrolyzed by enzymes attached to the outer surface of the intestinal epithelial cells ... 

Prenylation (covalent attachment of an isoprenoid see Fig. 27-30) is a common mechanism by which proteins are anchored to the inner surface of cellular membranes in mammals (see Fig. 11-14). In some of these proteins the attached lipid is the 15-carbon farnesyl group others have the 20-carbon geranylgeranyl group. Different enzymes attach the two types of lipids. It is possible that prenylation reactions target proteins to different membranes, depending on which lipid is attached. Protein prenylation is another important role for the isoprene derivatives of the pathway to cholesterol. 

The enzyme attached to antibody 2 is critical for quantitative analysis. Figure 19-14 shows two ways in which the enzyme can be used. The enzyme can transform a colorless reactant into a colored product. Because one enzyme molecule catalyzes the same reaction many times, many molecules of colored product are created for each analyte molecule. The enzyme thereby amplifies the signal in the chemical analysis. The higher the concentration of analyte in the original unknown, the more enzyme is bound and the greater the extent of the enzyme-catalyzed reaction. Alternatively, the enzyme can convert a nonfluorescent reactant into a fluorescent product. Colorimetric and fluorometric enzyme-linked immunosorbent assays are sensitive to less than a nanogram of analyte. Pregnancy tests are based on the immunoassay of a placental protein in urine. 

M. D. Lilly and A. K. Sharp, Kinetics of enzymes attached to water-insoluble polymers, Chem. Engineer, 1968, 21k, CE12-CE18. 

Another way considered as of biomimetic inspiration and that was shown to be efficient for enzyme attachment, it consists in using the very strong and specific interaction of the small protein avidin for the biotin [61,62]. The tetrameric structure of avidin permits itself to interact with four different molecules of biotin at the same time. Various proteins and enzyme could be easily biotinylated, and this mode of enzyme grafting has already been used for electrodes production as well as for membranes made up of conducting fibers. 

For enzyme attachment to the silicon microreactor tested, a layer-by-layer technique was employed to build a multilayer system of polyions and enzyme. Deposition of multilayers was accomplished by alternating positively and negatively charged layers of polydimethyldiallyl ammonium chloride (PDDA) and polystyrene sulfonate (PSS), respectively, to which was attached urease enzyme. After depositing in succession three layers of PDDA, PSS, and PDDA, three layers of urease enzyme were alternately deposited with three layers of PDDA. The resulting architecture is described as follows ... 

ELISA can be used to quantify the amount of a specific protein antigen in a sample. The antibody is bound to an inert polymer support, then exposed to the sample. Unbound protein is washed away and a second antibody that reacts with the antigen at a different epitope is added. The second antibody used is one that has an enzyme attached to it that converts a colorless or nonfluorescent substrate into a colored or fluorescent product. The amount of second antibody bound, and hence the amount of protein antigen present in the original sample, is determined by quantification of the intensity of color or fluorescence produced. 

When displaying an enzyme on a phage surface, evaluation of the level of functional enzymes attached to the phage particle is generally recommended prior to mutagenesis and selection. This level is equal to the ratio between the enzyme and the phage concentrations. 

The GT-B fold family includes most prokaryotic enzymes that produce secondary metabolites, like the antibiotics streptomycin, oleandomycin (Fig. 1) and vancomycin, and important bacterial cell wall precursors. It is also predicted to contain the vitally important 0-GlcNAc transferase that modifies many nuclear and cytoplasmic proteins and influences gene transcription. The first glycosyltransferase structure reported in 1994 was for the GT-B fold enzyme, P-glucosyltransferase (BGT) from bacteriophage T4 (22). This enzyme attaches glucose to modified... 

Monoamine oxidase, which exists in two distinct forms, referred to as MAO A and MAO B, is one of the enzymes responsible for the degradation of biologically important amines. Compounds that block the catalytic action of MAO A, which is selective for the degradation of norepinephrine and serotinin, have antidepressant effects whereas compounds that inhibit MAO B, which degrades dopamine in the brain, are useful for treating Parkinson s disease [190, 191]. Both MAO A and MAO B contain flavin co-enzyme attached at the 8-a-position to an enzyme-active cysteine residue (54). A one-electron transfer mechanism (Scheme 15) for the oxidations catalyzed by MAO was first proposed by Silverman [192] and Krantz [193,194]. 

A small fraction of the polypeptides contacting the brush border of the small intestine is hydrolyzed by membrane-bound enzymes attached to the outside of the enteroey te. Although a variety of peptidases are bound to the brush border, the most abundant is amlnopeptidase N, which catalyzes the hydrolysis of amino... 

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