The principles of enzyme stabilization IV. Modification of ‘key’ functional groups in the tertiary structure of proteins

Торчилин, В. П.; Maksimenko, A. V.; Смирнов, В. Н.; Березин, И.В.; Klibanov, Alexander M.; Martínek, Karel

doi:10.1016/0005-2744(79)90165-7

Cited by 67 publications

(25 citation statements)

References 21 publications

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“…At temperatures lower than that necessary for the transition E, * E, to proceed, thermoinactivation of a-chymotrypsin is assumed to be caused by irreversible conformational changes f25, 331. This assumption is supported by the fact that at temperatures lower than 50°C, the enzyme is stabilized by salting-out additives [4]. This stabilization is explained from the assumption [2, 41 that, in the presence of salting-out compounds, the protein molecule becomes much more rigid and less sensitive to unfolding.…”

Section: Mechanisms Of the Inactivation Of Conformations E And Ementioning

confidence: 99%

“…This parameter characterizes the effect of a solute present in a concentration cs, on the solubility, S, of a model compound as compared to the solubility of the same model compound in pure water, So: Thermoinactivation of a-chymotrypsin at temperatures below 55 "C proceeds according to the mechanism of 'incorrect refolding' [20, 251. According to this mechanism, protein unfolds on heating and in the course of the subsequent cooling refolds into a 'non-native' catalytically inactive conformation, in which it stays for purely kinetic reasons. The idea used previously for stabilization of a-chymotrypsin against such inactivation was to suppress the initial unfolding of the enzyme molecule by salting-out ions [4]. The value of the stabilization effect increased with an increase of saltingout capacity of salts [41.…”

mentioning

confidence: 99%

“…Surprisingly, salting-in additives stabilized a-chymotrypsin to some extent at moderate temperatures (45-55°C) as well. This fact cannot be explained, however, by the concept of the stabilization of the native folded form [4]. Moreover, at higher temperatures stabilization of a-chymotrypsin [3] and other enzymes [2,26] in solutions of urea and other salting-in compounds is even more pronounced.…”

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confidence: 99%

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Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution

Levitsky¹,

Panova²,

Mozhaev³

1994

European Journal of Biochemistry

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A correlation between the stability of a-chymotrypsin against irreversible thermal inactivation at high temperatures (long-term stability) and the coefficient of Setchenov equation as a measure of salting-idout efficiency of solutes in the Hofmeister series has been found. An increase in the concentration of salting-in solutes (KSCN, urea, guanidinium chloride, formamide) leads to a manyfold decrease of the inactivation rate of the enzyme. In contrast, addition of salting-out solutes has a small effect on the long-term stability of a-chymotrypsin at high temperatures. The effects of solutes are additive with respect to their salting-idout capacities ; the stabilizing action of the solutes is determined by the calculated Setchenov coefficient of solution. The correlation is explained by a solute-driven shift of the conformational equilibrium between the 'low-temperature' native and the 'high-temperature' denatured forms of the enzyme within the range of the kinetic scheme put forward in the preceding paper in this journal : irreversible inactivation of the high-temperature form proceeds much more slowly compared with the low-temperature form.a-Chymotrypsin is one of the favorite subjects for studies of the mechanisms of protein stability [l-41 and stabilization [5] (and references therein). Nevertheless, the influence of low-molecular-mass compounds on irreversible thermoinactivation of this enzyme (long-term stability) has not been studied at high temperatures (60-80°C). Such studies may provide a better practical application of enzymes because it is often necessary to preserve an active enzyme from inactivation during treatment at high temperatures. The preparation of protease-containing detergents, sterilization of medicines and some other biotechnological processes which include prolonged heating can be mentioned as examples [6]. On the other hand, even at lower temperatures (4O-5O0C), the effects on proteins of urea and guanidinium chloride (GdmC1) [7-91, salts [lo-121, organic solvents [13-151 and other solutes are very different and cannot be described easily by a general theory (for one of the best recent attempts, see [16]). Thus, a better understanding of denaturation at high temperatures may be welcomed from a theoretical point of view [17].The principal possibility of regulating long-term stability by solutes may be based on the idea that enzyme inactivation is a multi-step process often involving the reversible unfolding of the protein molecule [18-201. If in the folded native state and in the unfolded reversibly denatured state an enzyme is inactivated at different rates, one may try to control inactivation by shifting the conformational equilibrium between these states. The equilibrium constant that governs the reversible unfolding of proteins is influenced significantly by low-molecular-mass additives [14, 211. An ability of ions to favor either the folded or the unfolded state is determined qualitatively by their position in the Hofmeister series, on condition that ions do not preferentially ...

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Section: Mechanisms Of the Inactivation Of Conformations E And Ementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution

Levitsky¹,

Panova²,

Mozhaev³

1994

European Journal of Biochemistry

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“…2). As usually done [29,44], we define the stabilizing effect as a ratio of the first-order rate constants of thermoinactivation, observed for the more stable fractions (see Fig. 2) of the native and modified trypsin preparations (knat/kmOd).…”

Section: Thermoinactivation Of Modifiedpreparations Of Trypsinmentioning

confidence: 99%

Protein stabilization via hydrophilization

Mozhaev

Šikšnis

Melik‐Nubarov

et al. 1988

European Journal of Biochemistry

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This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach.1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one.2. a-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxylic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of cr-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65 -98 "C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date.Applications of enzymatic catalysis in biotechnology, fine organic synthesis, analysis, medicine, and other areas are often hindered because many enzymes, if isolated from their natural environment in vivo, become unstable and rapidly inactivate (denature); for review, see [l, 21. For these reasons the problem of enzyme stabilization has received considerable attention in recent years [3 -81.The analysis of the structure-stability relationships in the protein leads us to the conclusion [S] that the structure of proteins is not optimal with regard to their stability. There are still some reverses for stabilization which are used by nature for the production of extremely stable proteins, such as, for example, enzymes from thermophilic microorganisms ; for review, see [S, 91. Reserves of such a kind may be useful for artificial stabilization of enzymes. We shall discuss here, from this viewpoint, how to improve the protein structure in order to make enzymes more stable.According to X-ray crystallographic data, about one half of the surface area of proteins is occupied by nonpolar amino Corre2pondence to K. Martinek, Ustav OrganickC Chemie a Biochemie, Ceskoslovenska Akademie Vtid, Flemingovo nimEsti 2, CS-166-10 Praha, CzechoslovakiaThe authors wish to dedicate this paper to the memory of their teacher and friend. Professor...

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“…Por ej. El agente entrecruzante debe tener una longitud apropiada (Torchilin et al, 1979), y cuando se usan reactivos homo-bifuncionales, podría generarse una fuerte Rev. Acad.…”

Section: Modificación Química De Enzimasunclassified

Modificación Química en Fase Sólida de Lipasa B de Candida antarctica para mejorar sus propiedades de actividad, estabilidad y enantioselectividad

Torres-Sáez

2014

Rev. Acad. Colomb. Cienc. Ex. Fis. Nat.

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ResumenEn este trabajo, se llevó a cabo modificaciones químicas de preparaciones de lipasa B de Candida antarctica (CALB) inmovilizadas en soportes de octil-agarosa, agarosa-Bromuro Cianógeno-(BrCN) y Eupergit C usando diferentes compuestos químicos, por ej. Etilendiamina (EDA), anhídrido succínico (SA) y ácido 2,4,6-trinitrobencenosulfónico (TNBS). Estas modificaciones de la superficie de la enzima causó cambios en las propiedades tales como carga neta (punto isoeléctrico o balance de grupos catiónicos/aniónicos) o hidrofobicidad (solubilidad), y demostraron ser métodos prácticos para mejorar el funcionamiento del biocatalizador (estabilidad, actividad y enantioselectividad). Estas alteraciones en las propiedades de la enzima por modificación química podría ser debida a cambios en la estructura de la forma activa de CALB. De esta manera, la modificación química en fase sólida de lipasas inmovilizadas podría convertirse en una herramienta poderosa en el diseño de librerías de lipasas con propiedades muy diferentes.Palabras claves: Lipasa, Candida antarctica B, enzimas, modificación química. Chemical Modification in Solid Phase Chemistry of Lipase B from Candida antarctica for improving its properties of activity, stability and enantioselectivity AbstractIn this work, it was carried out chemical modifications of Candida antarctica lipase B (CALB) preparations immobilized on octyl-agarose, BrCN-agarose and Eupergit-C supports using different chemical compounds, e.g. ethylenediamine (EDA), succinic anhydride (SA) and 2,4,6-trinitrobenzensulfonic acid (TNBS). These modifications of the enzyme surface caused changes in physical properties such as net charge (isoelectric point or balance of cationic/anionic groups) or hydrophobicity (solubility), and proved to be practical methods to enhance the biocatalyst performance (stability, activity and enantio-selectivity). These alterations in enzyme properties by chemical modification should be due to changes in the structure of the active form of CALB. Therefore, solid phase chemical modification of immobilized lipases may become a powerful tool in the design of lipase libraries with very different properties.Key words: Lipase, Candida antarctica B, enzymes, chemical modification. Correspondencia:Rodrigo Gonzalo Torres-Sáez, rtorres@uis.edu.co Recibido: 6 de mayo de 2014 Aceptado: 16 de novimbre de 2014 Ciencias químicas IntroduciónLas enzimas son biocatalizadores que llevan a cabo diversos procesos químicos bajo condiciones suaves de reacción. Sin embargo, las enzimas se han seleccionado a través de procesos naturales de evolución, lo cual ha generado que la gran mayoría operen generalmente en condiciones ambientales suaves de reacción. Esta situación ha limitado mucho su aplicación en procesos industriales que utilizan condiciones drásticas de reacción. Por esta razón, se requiere la estabilización funcional de las enzimas en condiciones diferentes a las fisiológicas, de manera de ampliar su espectro de acción en procesos de transformación de sustratos que requieren co...

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The principles of enzyme stabilization IV. Modification of ‘key’ functional groups in the tertiary structure of proteins

Cited by 67 publications

References 21 publications

Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution

Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution

Protein stabilization via hydrophilization

Modificación Química en Fase Sólida de Lipasa B de Candida antarctica para mejorar sus propiedades de actividad, estabilidad y enantioselectividad

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