Modifiers of enzymes

The retention of 1,2-dibromoethane in tissues and body fluids can be altered by concurrent exposure to modifiers of enzyme activity, such as disulfiram (Plotnick et al. 1979). The concentration of radiolabeled 1,2-dibromoethane in the liver, kidneys, spleen, testes, and brain increased significantly in rats fed disulfiram in the diet for 12 days before an oral dose of 15 mg C-1,2- dibromoethane/kg compared with rats not fed disulfiram. Disulfiram, an inhibitor of P-450 metabolism (via action on acetaldehyde dehydrogenase), was found to increase the uptake of C into liver nuclei. These observations correlate well with the results of chronic studies (Wong et al. 1982) that demonstrated enhanced tumorigenic effects in the liver and testes following combined 1,2-dibromoethane and disulfiram exposure. 

Textile finishing includes various efforts to improve the properties of textile fabrics, whether for apparel, home, or other end uses. In particular, these processes are directed toward modifying either the fiber characteristics themselves or the gross textile end properties. Such modifications may be chemical or mechanical in nature. One modification that is not covered in this article relates to the dyeing of textiles and the dyestuffs employed for fibers however, areas that involve chemical finishing designed to modify the normal dye receptivity and the growing use of enzyme treatments are included. 

Fig. 3. Detail of enzyme-modified quartz radio crystal used in a piezoelectric biocatalytic biosensor.

Other immobilization methods are based on chemical and physical binding to soHd supports, eg, polysaccharides, polymers, glass, and other chemically and physically stable materials, which are usually modified with functional groups such as amine, carboxy, epoxy, phenyl, or alkane to enable covalent coupling to amino acid side chains on the enzyme surface. These supports may be macroporous, with pore diameters in the range 30—300 nm, to facihtate accommodation of enzyme within a support particle. Ionic and nonionic adsorption to macroporous supports is a gentle, simple, and often efficient method. Use of powdered enzyme, or enzyme precipitated on inert supports, may be adequate for use in nonaqueous media. Entrapment in polysaccharide/polymer gels is used for both cells and isolated enzymes. 

Enzymes in Pulp and Paper Production. Enzyme-modified starch has been used for adhesives to strengthen paper base and for surface coating. Developments since the late 1980s of further uses of enzymes in papermaking include pitch control and bleach boosting, (see Paper Pulp). 

FIGURE 18.5 Schematic representation of types of multienzyme systems carrying out a metabolic pathway (a) Physically separate, soluble enzymes with diffusing intermediates, (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes Ei to E5 before product is released. No intermediates are free to diffuse away, (c) A membrane-bound multienzyme system. 

Many of these have been demonstrated with a range of antibiotics and antibiotic precursors, although relatively few have been applied commercially. We have included a list of published examples in the form of an Appendix at the end of this chapter. We do not expect you to remember the details of this Appendix. It has been included as an potential for illustration of the potential to use enzymes to modify organic molecules like antibiotics. Usin9 It should be anticipated that, as enzyme technology develops and the search for new enzymes antibiotics continues, an increasing number of enzyme-based transformation will find commercial application. 

The enzymatic transformation of natural products is by for file most attractive option. In this approach, it can be envisaged that sterols, which are relatively abundant, may be selectively modified to produce desired products. Hie diversity of enzyme activities, their reaction specificity, regiospecificity and stereospedfidty are all features which could contribute to carrying out the desired changes. This does not mean, however, that transformations using enzyme systems are simple. Nevertheless, biotransformations have become of vital importance in the production of steroids. 

Another type of NR crosstalk, which has only recently been recognized, is the so-called nongenomic actions of several receptors that induce very rapid cellular effects. Effectively, evidence has accumulated over several decades that steroid receptors may have a role that does not require their transcriptional activation, such as modifying the activity of enzymes and ion channels. While the effects of steroids that are mediated by the modulation of gene expression do occur with a time lag of hours, steroids can induce an increase in several second messengers such as inositol triphosphate, cAMP, Ca2+, and the activation of MARK and PI3 kinase within seconds or minutes. Many mechanistic details of these nongenomic phenomena remain poorly understood. Notably, controversy still exists as to the identity of the receptors that initiate the non-genomic steroid actions. However, it now appears that at least some of the reported effects can be attributed to the same steroid receptors that are known as NRs. 

Small tfbiquitin-like modifier represents a family of evolutionary conserved proteins that are distantly related in amino-acid sequence to ubiquitin, but share the same structural folding with ubiquitin proteins. SUMO proteins are covalently conjugated to protein substrates by an isopeptide bond through their carboxyl termini. SUMO addition to lysine residues of target proteins, termed SUMOylation, mediates post-transla-tional modification and requires a set of enzymes that are distinct from those that act on ubiquitin. SUMOylation regulates the activity of a variety of tar get proteins including transcription factors. 

In principle, numerous reports have detailed the possibility to modify an enzyme to carry out a different type of reaction than that of its attributed function, and the possibility to modify the cofactor of the enzyme has been well explored [8,10]. Recently, the possibility to directly observe reactions, normally not catalyzed by an enzyme when choosing a modified substrate, has been reported under the concept of catalytic promiscuity [9], a phenomenon that is believed to be involved in the appearance of new enzyme functions during the course of evolution [23]. A recent example of catalytic promiscuity of possible interest for novel biotransformations concerns the discovery that mutation of the nucleophilic serine residue in the active site of Candida antarctica lipase B produces a mutant (SerlOSAla) capable of efficiently catalyzing the Michael addition of acetyl acetone to methyl vinyl ketone [24]. The oxyanion hole is believed to be complex and activate the carbonyl group of the electrophile, while the histidine nucleophile takes care of generating the acetyl acetonate anion by deprotonation of the carbon (Figure 3.5). 

Inhibitors of the catalytic activities of enzymes provide both pharmacologic agents and research tools for study of the mechanism of enzyme action. Inhibitors can be classified based upon their site of action on the enzyme, on whether or not they chemically modify the enzyme, or on the kinetic parameters they influence. KineticaUy, we distinguish two classes of inhibitors based upon whether raising the substrate concentration does or does not overcome the inhibition. 

Lipogenesis is regulated at the acetyl-CoA carboxylase step by allosteric modifiers, phosphorylation/de-phosphorylation, and induction and repression of enzyme synthesis. Citrate activates the enzyme, and long-chain acyl-CoA inhibits its activity. Insulin activates acetyl-CoA carboxylase whereas glucagon and epinephrine have opposite actions. 

The apparent reliance of enzyme activation on phosphorylation and intracellular Ca + gives a clue as to how the rate of 5-HT synthesis might be coupled to its impulse-evoked release. Certainly, the impulse-induced increase in intracellular Ca +, and/or activation of the G protein-coupled receptors that govern synthesis of cAMP, could modify the activity of tryptophan hydroxylase. Indeed, this could explain why activation of either somal 5-HTia autoreceptors in the Raphe nuclei (which depress the firing rate of 5-HT neurons) or terminal 5-HTib autoreceptors (which depress 5-HT release) can reduce the production of cAMP and attenuate 5-HT synthesis. 

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