Enzymes and proteins

So far, it has been established from in vitro studies that the enzyme undergoes phosphorylation, a process that changes the conformation of the enzyme protein and leads to an increase in its activity. This involves Ca +/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase which suggests a role for both intracellular Ca + and enzyme phosphorylation in the activation of tryptophan hydroxylase. Indeed, enzyme purified from brain tissue innervated by rostrally projecting 5-HT neurons, that have been stimulated previously in vivo, has a higher activity than that derived from unstimulated tissue but this increase rests on the presence of Ca + in the incubation medium. Also, when incubated under conditions which are appropriate for phosphorylation, the of tryptophan hydroxylase for its co-factor and substrate is reduced whereas its Fmax is increased unless the enzyme is purified from neurons that have been stimulated in vivo, suggesting that the neuronal depolarisation in vivo has already caused phosphorylation of the enzyme. This is supported by evidence that the enzyme activation caused by neuronal depolarisation is blocked by a Ca +/calmodulin protein kinase inhibitor. However, whereas depolarisation... 

The number of enzymes and functional proteins that are reportedly regulated by S-nitrosation is on the rise. For example, a search of PUBMED with the key word S-nitrosation revealed some 70 reports of in vitro regulation of enzymes, proteins and cellular processes that are affected by S-nitrosation. Some of these processes that have been well characterized include, nuclear regulatory proteins the NMDA receptor and the ertrocyte anion exchange protein 1 (AE1) (see review by Gaston, 2003). 

It is of interest in this connection that Steam,26 in a discussion of enzyme kinetics from the point of view of statistical mechanics and quantum mechanics, regards interaction between a dipole (as part of the enzyme protein) and the reacting groups of the substrate, e.g., C—0, resulting in a redistribution of charge within the C—0 bond, as a more rational mechanism of activation than the loosening of the bond by distortion. 

Allosteric regulators bind to the target enzyme in a non-covalent manner. An entirely different enzyme control mechanism is covalent modification. Here, the conformation of the enzyme protein, and thereby its activity, is changed by the... 

Some inhibitors interact very slowly with the enzyme protein, and onset of inhibition thus exhibits time-dependence. These inhibitors are generally referred to as slow-binding inhibitors, and as slow tight-binding inhihitors if the potency of inhibition is extremely high. Analysis of these inhibitory mechanisms is complex because binding and dissociation rate constants may be determined in addition to values. Indeed, a complete analysis may require extensive use of specialized computer software, and the complexities of such analyses preclude their discussion in this chapter. However, the reader is directed to several publications from Morrison s laboratory if a slow-binding mechanism is suspected for an inhibitor of interest (Morrison, 1982 Morrison and Stone, 1985 Sculley and Morrison, 1986 Morrison and Walsh, 1988). 

Most ultrasonic experiments are carried out in temperature controlled systems to ensure that isothermal conditions are maintained. Even a small general increase in microbial temperature can influence both the active and passive transport systems of the cell membrane/wall and this in turn may lead to an increased uptake of compounds. If the temperature is not controlled then sonication could result in a large temperature increase which will lead to the denaturation (deactivation) of enzymes, proteins and other cellular components present within the microorganism [7]. 

About 90 g of protein is ingested each day by an adult on a typical Western diet and a similar amount enters the intestine from the secretion of enzymes, proteins and from sloughed epithelial cells (Chapter 8). The proteins are hydrolysed by proteolytic enzymes. 

C) Induction of drug-metabolizing enzymes frequently requires the synthesis of new enzyme protein and thus may not occur immediately upon introduction of the inducing agent. 

Metabolic control can be understood to some extent by focusing attention on those enzymes that catalyze rate-limiting steps in a reaction sequence. Such pacemaker enzymes1-4 are often involved in reactions that determine the overall respiration rate of a cell, reactions that initiate major metabolic sequences, or reactions that initiate branch pathways in metabolism. Often the first step in a unique biosynthetic pathway for a compound acts as the pacemaker reaction. Such a reaction may be described as the committed step of the pathway. It usually proceeds with a large decrease in Gibbs energy and tends to be tightly controlled. Both the rate of synthesis of the enzyme protein and the activity of the enzyme, once it is formed, may be inhibited by feedback inhibition which occurs when an end product of a biosynthetic pathway accumulates... 

The reversible inhibitor. This inhibitor can come off of the enzyme protein and thus can be reversed. 

Ozeki, Y., Komamine, A. Tanaka, Y. (1990). Induction and repression of phenylalanine ammonia-lyase and chalcone synthase enzyme proteins and mRNAs in carrot cell suspension cultures regulated by 2,4-D. Physiologia Plantarum 78, 400-8. 

When working with enzymes, proteins and other biological molecules it is often crucial to buffer the pH of the solution in order to avoid denaturation (loss of activity) of the component of interest (see Topic C3). Numerous buffers are used in laboratories for this purpose. One of the commonest is tris(hydroxy-methyl)aminomethane or TRIS which has a pK of 8.08. 

We have discussed both target receptors and pharmacokinetics in this book. Protein manufacture is under direct genetic control, and two factors are of particular relevance here. First, the precise structure and function of protein macromolecules (receptors) targeted by a specific drug molecule will vary in different individuals. Since the structure and function of the protein are directly related to how the drug molecule will interact with that protein, individuals responses to the drug will vary. Second, there are genetic variations in metabolic enzymes (proteins) and hence metabolism. Both of these processes fall neatly into the domain of pharmacoproteomics (see Section 14.8). 

Metals can directly or indirectly interfere with cellular components and structures. Their effects on enzyme proteins and biomembranes were discussed extensively. Depending on the enzyme studied, metals either inhibit or stimulate its activity. They... 

The non-adiabatic long-range electron transfer (LRET) has been proven to be one of the key stages of many processes in enzymes, proteins and model systems. Therefore, theoretical calculation and experimental determination of the resonance integral (V) and its dependence on the distance between donor and acceptor centers appears to be a fundamental problem. 

The reaction mixture contained Tris-Q (pH 8.0), MgCk, glutathione as the acceptor, [35S]PAPS, and protein. Samples were withdrawn, and the reaction was terminated by forcing the sample through a microfilter at 6 bar under nitrogen. The filter retained the enzyme protein, and the nucleotides were recovered. 

Metabolic Control of CoA Synthesis Pantothenate kinase is rate-limiting for the synthesis of CoA, and both regulation of the activity of the existing enzyme protein and changes in its synthesis are important in the control of intracellular concentrations of CoA (Rock et al., 2000). The enzyme has a low Km compared with the normal intracellular concentration of pantothenic acid and is thus insensitive to the availability of substrate, even in deflciency. 

In fact, the enzyme-inhibitor interaction in itself is a chain of complex processes for the inhibitor molecule, including a number of desolvations, collisions with nonspecific sites on the enzyme protein, and resolvations before reaching the specific inhibition site. The complexity is not at all less than those considered for the transport and drug-receptor interaction processes of cytokinins. The situation is analogous to that a set of every rational number between zero and one corresponds in a one-to-one... 

The greatest variety of polyelectrolytes is found in nature. The role of electric charges is essential for the proper functioning of nucleic acids, the numerous enzymes, proteins and polysacchardies. The fundamental role of polyelectrolytes in all living processes is unquestionable. 

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