Enzymes, unfolding

The immobilization procedure may alter the behavior of the enzyme (compared to its behavior in homogeneous solution). For example, the apparent parameters of an enzyme-catalyzed reaction (optimum temperature or pH, maximum velocity, etc.) may all be changed when an enzyme is immobilized. Improved stability may also accrue from the minimization of enzyme unfolding associated with the immobilization step. Overall, careful engineering of the enzyme microenvironment (on the surface) can be used to greatly enhance the sensor performance. More information on enzyme immobilization schemes can be found in several reviews (7,8). 

Fig. 14.20. Schematic representation of the adsorption process of glucose oxidase (GO) (a) The potential is much more positive than the potential of zero charge, (b) The potential is close to the pzc. (c) The final stage when the enzyme unfolded. (Reprinted from A. Szucs, G. D. Hitchens and J. O M. Bockris, J. Electrochem. Soc. 136(12) 3748, Fig. 13, 1989. Reproduced by permission of the Electrochemical Society, Inc.)...

Recovered activity and immobilization yield were calculated, and results are listed in Table 1. It can be observed that recovered activity increased when the initial enzyme concentration in the supernatant (Eq) was increased to 60 and 90 U/ml, but it remained almost constant for the other concentrations studied. Immobilization yield, on the other hand, decreased when high concentrations of enzyme were used (60 to 150 U/ml, compared to 30-40 U/ml). When Ef,=9Q U/ml, the highest value of recovered activity was obtained, which suggests that protein molecules are probably immobilized at close proximity to each other, which may prevent deactivation caused by enzyme unfolding by covering the support surface. In other words, when enzyme load was increased, more enzyme molecules were immobilized and less area of the support is available for lipase to spread itself, which may prevent loss in activity [31]. 

Goan, K.E., Maltby, D.A., Burlingame, A.L., and Shoichet, B.K (2009) Promiscuous aggregate-based inhibitors promote enzyme unfolding. Journal of Medicinal Chemistry, 52, 2067-2075. 

Exposure to high temperatures poses a danger to living organisms because essential proteins and enzymes unfold, a process called denaturation. When this happens, the compounds no longer mate with complementary shape receptors or chemicals, and normal biological activities cease. 

The interaction between an enzyme and a material surface can either increase or deaease its catalytic activity, and the actual outcome depends on the nature of the enzyme, the nature of the material surfaces and the microenvironment produced by their interaction. Changes in enzyme conformation caused by immobilization may alter its interactions with different substrates and its stability with respect to solvent, temperature, and pH. It is therefore highly valuable to understand how an enzyme unfolds in order to choose an adequate support for... 

When 6 moles of the substrate analog PALA combine with the enzyme ACTase, two things happen at the same time. The enzyme T unfolds to a more active form R... 

Use all three equations to find the enthalpies of binding and of unfolding for this enzyme. 

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. 

Enzymes assist formation of proper disulfide bonds during folding Isomerization of proline residues can be a rate-limiting step in protein folding Proteins can fold or unfold inside chaperonins GroEL is a cylindrical structure with a... 

Denaturation is accompanied by changes in both physical and biological properties. Solubility is drastically decreased, as occurs when egg white is cooked and the albumins unfold and coagulate. Most enzymes also lose all catalytic activity when denatured, since a precisely defined tertiary structure is required for their action. Although most denaturation is irreversible, some cases are known where spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs. Renaturation is accompanied by a full recovery of biological activity. 

The reason that the caseins, which constitute nearly 80% of bovine milk, are unfolded in their native states appears to be to facilitate digestion, since the open rheomorphic structures allow rapid and extensive degradation to smaller peptides by proteolytic enzymes. The natively unfolded structures of many cereal proteins may serve an analogous purpose since they provide nutrition for seedlings. The physiological function of the synucleins in the brain is as yet unclear, but tau is known to promote and stabilize the assembly of microtubules. 

The molecular folding of the backbone chain of the enzyme, as well as the distribution and content of amino acids [71] plays a decisive role in determining the characteristic specificity of an enzyme with regard to its reactions. This folding is markedly affected by temperature, for example. As the temperature rises, the chain gradually unfolds until a point is reached at which the enzyme becomes denatured and the catalytic activity is lost. 

Fig. 9.4 The enzyme RNase can be unfolded by reduction agents (urea and mercaptoethanol). Oxidative removal of the reduction agents causes the molecule to take up its three-dimensional structure again it regains almost its full enzymatic activity...

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